1

                        UNITED STATES OF AMERICA

                 DEPARTMENT OF HEALTH AND HUMAN SERVICES

                          PUBLIC HEALTH SERVICE

                      FOOD AND DRUG ADMINISTRATION

              CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

                    AND NATIONAL INSTITUTES OF HEALTH

              NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS
                                DISEASES 

                WARREN GRANT MAGNUSON CLINICAL CENTER NIH

                      GRANULOCYTES FOR TRANSFUSION:
                    RESEARCH AND CLINICAL EXPERIENCE

                                WORKSHOP

                                Friday, 

                           September 11, 1998

                The workshop took place in the Jack Masur
          Auditorium, National Institutes of Health, 9000
          Rockville Pike, Bethesda, MD 20892,  at 8:00 a.m.,
          Liana Harvath, Ph.D., Chair, presiding. 

          PRESENT:

                KATHRYN C. ZOON, Ph.D., Director, CBER
                JOHN GALLIN, M.D., Director, WGMCC
                LIANA HARVATH, Ph.D., Chair
                DAVID STRONCEK, M.D., Moderator

                DOUGLAS ADKINS, M.D., Speaker
                DANIEL AMBRUSO, M.D., Speaker
                DAVID DALE, M.D., Speaker
                JOSE-LUIS DIAZ, Ph.D., Speaker
                THOMAS LANE, M.D., Speaker
                SUSAN LEITMAN, M.D., Speaker
                CONRAD LILES, M.D., Ph.D., Speaker

                THOMAS PRICE, M.D., Speaker

          ALSO PRESENT:

                JAY EPSTEIN, M.D.

                                                                          2

                                 AGENDA

          Welcome/Introduction
            Kathryn C.Zoon, Ph.D., Director CBER/FDA  . . .  3
            John Gallin, M.D., Director, WGMCC/NIAID  . . .  5
            
          SESSION I 
            Moderator Liana Harvath, Ph.D.  . . . . . . . .  7

          Historical Perspective & Clinical Trial Considerations
            David Dale, M.D.  . . . . . . . . . . . . . . .  9

          Cytokine Administration to Normal Granulocyte Donors
            Thomas Price, M.D.  . . . . . . . . . . . . . . 47

          Functional Properties of Granulocytes from Normal

          Donors after G-CSF Administration
            Daniel Ambruso, M.D.  . . . . . . . . . . . . . 71

          Cytokine Mobilized Granulocytes:  Assessment of 
          Efficacy
            Susan Leitman, M.D. . . . . . . . . . . . . . . 91

          Panel Discussion  . . . . . . . . . . . . . . .  120

          SESSION II 
            Moderator David Stroncek, M.D.  . . . . . . .  154

          Product Evaluation
            David Adkins, M.D.  . . . . . . . . . . . . .  154

          Storage Considerations
            Thomas Lane, M.D. . . . . . . . . . . . . . .  175

          In vitro Assays Predictive of Product Function
            Conrad Liles, M.D., Ph.D. . . . . . . . . . .  197

          Panel Discussion  . . . . . . . . . . . . . . .  216

          Presentation of Submitted Abstracts 
            David Stroncek, M.D.  . . . . . . . . . . . .  229
            Dr. Diaz  . . . . . . . . . . . . . . . . . .  242

            

                                                                          3

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

     2                                             (8:08 a.m.)

     3                CHAIRPERSON HARVATH:  Good morning.  I

     4    think we will get started in the spirit of trying to

     5    keep on time.  On behalf of the Steering Committee for

     6    this workshop, it is a pleasure to introduce two very

     7    distinguished individuals, Director for the Center of

     8    Biologics Evaluation and Research, Dr. Kathryn Zoon,

     9    who will give welcoming remarks, and Dr. John Gallin,

    10    who in this audience really needs no introduction

    11    since his career in the granulocyte field has made

    12    enormous contributions to this field.  Dr. Gallin will

    13    be speaking on behalf as the Director of the Clinical

    14    Center as well as making remarks on behalf of NIAID. 

    15    Dr. Zoon?

    16                DR. ZOON:  Good morning.  It is a pleasure

    17    to be here and to open this important workshop with

    18    Dr. Gallin.  John and I have known each other many

    19    years, in our interferon days and working on various

    20    activities of interferon gamma and granulocytes.  I

    21    think there is lots of work to still be done and I am

    22    very anxious to hear the science today and understand

    23    where we are with the technology and where we need to

    24    go tomorrow.

    25                It is a pleasure to welcome you on behalf

                                                                          4

     1    of the Center for Biologics Evaluation and Research,

     2    which is one of the FDA centers that has oversight of

     3    blood and blood products.  This workshop today, I

     4    believe, will really help us focus on some very

     5    important issues.  The findings that the

     6    administration of cytokine, such as granulocyte colony

     7    stimulating factor and granulocyte macrophage colony

     8    stimulating factors to normal volunteers results in

     9    the peripheral mobilization of high concentrations of

    10    granulocytes have renewed the interest in the

    11    collection of granulocytes for transfusion.  I think

    12    all of us have become very familiar in the literature

    13    with both the effects of G and GM-CSF on this.  And

    14    while there was a lot of interest in granulocytes for

    15    transfusions -- this peaked back in the 1970's --

    16    there is now a renewed interest because of our new

    17    tools.  We are very interested in exploring both the

    18    efficacy parameters as well as the safety parameters

    19    associated with this.

    20                We are very interested, and of course

    21    there are many others interested, in the scientific

    22    and clinical experience with cytokine mobilized

    23    granulocyte transfusion products and the effects of

    24    the cytokine administration on normal donors.  This is

    25    clearly important with regard to the safety of the

                                                                          5

     1    patients and the donors, but also important in the

     2    quality of the products, in this case the granulocytes

     3    looking at their functional capabilities.  

     4                Our colleagues at the NIH are here today

     5    to hear what areas in this field need to be further

     6    explored and supported in the research area, and we

     7    really are very appreciative of doing this workshop

     8    today in a collaborative effort, because many of the

     9    fruits of the science and the research that do arise

    10    come to FDA for our review and evaluation, and it is

    11    very important that the Agency be very active in the

    12    science and understanding the science so that we can

    13    do the very best job at facilitating the review and

    14    access of these important products.

    15                This is the second workshop this week.  We

    16    had one yesterday on hematopoietic stem progenitor

    17    cells and this one today on granulocytes for

    18    transfusion, and we really appreciate the attendance

    19    today.  We feel this is a very important area.  We

    20    feel very strongly that we need the best scientific

    21    data in which to move forward, and I wish you a very

    22    good meeting and look forward to the fruits of this

    23    workshop.  Thank you very much.

    24                DR. GALLIN:  Well, normally I don't know

    25    that I would get up and be willing to share in two

                                                                          6

     1    introductions to one meeting, but for this meeting I

     2    couldn't resist.  And the reason is because of my own

     3    longstanding personal affection for the granulocytes,

     4    which are by my way of thinking the most beautiful

     5    cells in the body, and also because of the importance

     6    of granulocyte transfusions.  So on behalf of both the

     7    Warren Grant Magnuson Clinical Center and all the

     8    staff who work here as well as the National Institute

     9    of Allergy and Infectious diseases, both of which are

    10    places that I work, we welcome you here.

    11                Now my personal interest really goes back

    12    to the use of granulocyte transfusion in patients with

    13    granulocyte defects, particularly patients with

    14    chronic granulomatous disease and a few patients that

    15    we have seen in this building with neutrophil specific

    16    granule deficiency.  And over the last 25 years, we

    17    have been convinced, truly on anecdotal evidence, that

    18    there are some patients in whom granulocyte

    19    transfusions made a difference in helping them get

    20    over life-threatening infections.  But, of course,

    21    there is no proof that they work, and that has always

    22    been an issue.  And we have also been worried that

    23    maybe we were actually doing something bad or

    24    potentially bad.  Perhaps we always worried that maybe

    25    we would be precipitating ARDS in patients with

                                                                          7

     1    chronic granulomatous disease or that we would be

     2    alloimmunizing the patients to an extent that future

     3    bone marrow transplants or gene therapy might be

     4    compromised.

     5                Nonetheless, we continued to use them on

     6    occasion.  So I personally really look forward to the

     7    results of your deliberations today with the hope that

     8    you will come to some conclusions in terms of not only

     9    the correct approaches for mobilizing these cells and

    10    harvesting them and storing them, but also for

    11    designing some clinical trials that will answer some

    12    of the definitive questions.  So have a great

    13    symposium.

    14                CHAIRPERSON HARVATH:  This is a great

    15    pleasure for me to be involved in helping to organize

    16    such a workshop, because my own area of research for

    17    the past 25 years also involves those beautiful cells

    18    of the body, the granulocytes.  

    19                In looking through our sort of cryptic

    20    files of workshops at the FDA, I came upon some

    21    transcripts of a workshop held in October of 1980 in

    22    Natcher Auditorium, and it was called the Conference

    23    on Leukopheresis Donor Safety.  And that was actually

    24    the last conference that FDA was involved in where

    25    there was a discussion of the collection of

                                                                          8

     1    granulocytes and leukocyte products, and the

     2    discussion at that time focused on donor safety issues

     3    when donors are given hetastarch or when they are

     4    given steroids for mobilization.  

     5                There were a number of participants in

     6    that workshop who were actually serving on the

     7    Steering Committee.  So what I would like to do is to

     8    in the next slide -- what I had is a list of people. 

     9    There should be two slides in that carousel.  The

    10    second slide -- there we go, thank you -- is to

    11    acknowledge and thank the members of the Steering

    12    Committee.  All of these people have distinguished

    13    themselves in the granulocyte field, and I would like

    14    to go through this list.  Dr. Daniel Ambruso from the

    15    University of Colorado and Bonfils Blood Center will

    16    be speaking to us;  Dr. David Dale from the University

    17    of Washington; Dr. John Gallin; Dr. Jeffrey

    18    McCullough, from the University of Minnesota, who

    19    unfortunately can't be here but who made major

    20    suggestions for the organization of this workshop; Dr.

    21    George Nemo from the Heart, Lung and Blood Institute;

    22    Dr. Daniel Rotrosen, who worked in this building with

    23    Dr. Gallin and Dr. Malik in neutrophil research; Dr.

    24    Ron Strauss from the University of Iowa, who is one of

    25    the pioneers in this field, who unfortunately can't be

                                                                          9

     1    here because he is giving a talk on granulocyte

     2    transfusions at the Pediatric Oncology Meetings today

     3    in Chicago; Dr. David Stroncek, who now works here at

     4    the NIH Clinical Center, and you will hear some of his

     5    work this afternoon; and finally, our colleague,

     6    Joseph Wilczek, who has served as the program

     7    coordinator in taking care of the laborious details

     8    that go into putting a conference like this together. 

     9                It is a great pleasure for me to have the

    10    opportunity to introduce the speakers in the morning

    11    session.  Dr. David Dale began his interest in

    12    granulocytes almost 30 years ago in this very

    13    building, where he was working as a clinical

    14    researcher, and he is now a professor of medicine and

    15    has had a very distinguished career in education and

    16    research in this field.  Dr. Dale is going to talk

    17    about the historical perspective and clinical trial

    18    considerations for granulocytes for transfusion.

    19                DR. DALE:  Well, thank you, Liana.  It is

    20    nice to be here.  I would say nice to be here again. 

    21    I think I last spoke about this topic in this room 25

    22    years ago, so it does bring back a lot of memories.

    23                If I can have the first slide, which is

    24    just a title slide.  I am going to talk this morning

    25    about really three things.  I am going to talk about

                                                                         10

     1    history.  That is always a dangerous thing to do.  I

     2    will mention a lot of names.  I may not mention

     3    everyone because of the time, but there have been many

     4    people involved over the years in studying this topic,

     5    many who have done other things in their lives and you

     6    will recognize them.  I am going to talk a bit about

     7    the effects of G-CSF in particular on neutrophil

     8    formation and function, again a part of the background

     9    for our overall discussions today.  And then finally

    10    talk about recent history, and that is the data that

    11    has led up, I think, in many ways.  And then finally

    12    to mention a little bit about clinical trial

    13    considerations.

    14                Our focus today is on the neutrophil,

    15    which my predecessors here this morning have described

    16    as beautiful, and they really are interesting cells. 

    17    Most oncologists see them on a laboratory slip as a

    18    number.  Hematologists may see them on a blood smear. 

    19    But they are truly interesting and beautiful cells. 

    20    Perhaps made more interesting and beautiful if you see

    21    them in a diagram like this, which shows some of the

    22    features of a neutrophil.  I won't dwell upon this

    23    today, but suffice it to say that there are many

    24    features of these cells that are regulated very

    25    tightly, and the cells that circulate in the

                                                                         11

     1    circulation are not always the same.  Infection, the

     2    administration of hematopoietic growth factors,

     3    diseases, many factors can change the characteristics

     4    of these cells.  The key features though are the

     5    surface of the cell, where the cell has receptors

     6    which allow it to interact with its environment, the

     7    granules of the cells, which are critical for the cell

     8    function, and then, of course, the nucleus, which

     9    allows us so easily to recognize a neutrophil in a

    10    blood smear.

    11                In terms of the formation of neutrophils,

    12    as a background statement I think it is very important

    13    to think about the kinetics.  Because the dynamics of

    14    how the body produces and how neutrophils are

    15    distributed in the blood and their ultimate fate have

    16    so much to do with the development of this field, both

    17    in the past and in the future.  Neutrophils are formed

    18    from hematopoietic stem cells and the steps of

    19    proliferation and differentiation and then maturation

    20    are very unique for these cells. The most unique

    21    feature compared to other blood cells is the storage

    22    in the marrow of a substantial portion of the body's

    23    total supply of the cells.  How much is it?  Well, it

    24    is probably 10 times the circulating supply or perhaps

    25    more depending upon where you draw the line.  But

                                                                         12

     1    suffice it to say it is a very large reserve, a

     2    reserve that is not there for monocytes or eosinophils

     3    or red cells or other of the hematopoietic cells.  And

     4    it is really this reserve supply of neutrophils in the

     5    marrow that are critical for the development of the

     6    whole concept of collecting neutrophils from normal

     7    donors for transfusion.  Neutrophils, as you will

     8    recall well, have a short blood lifespan, and in fact

     9    almost all of their function are in the tissues.  And

    10    although clinically over many years we have related

    11    susceptibility to infection to the number in the

    12    blood, it is actually the total body supply and the

    13    ability to deliver these cells to the tissues which is

    14    critical for the outcome in terms of the problem we

    15    are talking about.  

    16                And then finally as background, I will

    17    mention the process for the killing of organisms,

    18    represented by this pink cigar here, by a neutrophil

    19    is a complex process that we have unraveled in

    20    research supported here and in much done here as well

    21    as elsewhere around the world, to clarify the various

    22    processes involved in the dumping of myeloperoxidase

    23    into the phagocytic vacuole and the involvement of

    24    oxygen and oxygen derivatives in the actual killing of

    25    these organisms.  And it is this event, actually the

                                                                         13

     1    ability to kill organisms, that is why we are all

     2    healthy enough to be here today.  So it is vitally

     3    important not just for sick people but for people who

     4    regard themselves as healthy.

     5                Now critical finally for the understanding

     6    of this topic is the short lifespan or the rapid

     7    turnover of neutrophils.  This is illustrated here

     8    just by comparison with other blood cells.  Red cells

     9    having a lifespan of roughly three months or a

    10    turnover rate of 1 percent per day, platelets roughly

    11    a tenth of that in terms of their lifespan, and ten

    12    times as rapid turnover, but neutrophils are among the

    13    most rapidly turning over cells in the body.  In many

    14    audiences I have described it as just think of the

    15    fact that in your blood today, you have a whole fresh

    16    supply of neutrophils from what you had yesterday. 

    17    You have replaced all of those cells.  And if you go

    18    just a few days without a new supply, of course you

    19    are in trouble, as we all know.  

    20                Now in terms of thinking of history, this

    21    is actually an old slide, I think one that I showed

    22    here in a slight variation 25 years ago.  The problem

    23    of neutropenia has been one that has been recognized

    24    for almost the whole 20th Century.  The problem was

    25    actually recognized soon after the turn of the

                                                                         14

     1    century, with neutrophils being counted in blood

     2    smears over the few years before but really not very

     3    long.  But the treatment of neutropenia has

     4    languished.  This is basically our 1970's approach,

     5    that is, the use of antibiotics, and we still depend

     6    upon them to deal with the problem of neutropenia.  

     7    We had then in the 1970's a variety of agents to try

     8    to increase neutrophil production, I would say all

     9    very, very weak.  There were efforts to try to treat

    10    neutropenia with other factors such as removing the

    11    spleen in chronic states, but of course this was not

    12    applicable to acute states as occurs after

    13    chemotherapy.  And finally then, as now, a key concept

    14    in the treatment of neutropenia was an alternate

    15    supply, that is, to transfuse the cells.  

    16                In terms of the history of this idea, that

    17    is, the history of transfusing neutrophils to treat

    18    neutropenia, most reviewers of the topic would point

    19    to a study done in 1934, the height of the Depression,

    20    a study involving injection of buffy coat cells into

    21    patients, intramuscular injections, which were

    22    undoubtedly very painful and basically had no effect

    23    on the patients.  But it was a part of a desperate

    24    approach in the pre-antibiotic era to doing something

    25    about the problem of neutropenia.

                                                                         15

     1                Actually, the first important

     2    investigation in this area was conducted here by Dr.

     3    George Brecher and associates, working with Gene

     4    Cronkite, who was then at the Naval Medical Center,

     5    and that study, I think, was a landmark in terms of

     6    the building of the basic physiological concepts

     7    underlying this field.  What Dr. Brecher did -- I

     8    don't know, somebody in the room may know him -- I

     9    remember him from when I first came here, a really

    10    great man.  What he did were studies where donors,

    11    that is, dogs which had been made aplastic with

    12    radiation -- the donors were injected with turpentine

    13    to try to increase their counts and then the

    14    recipients were irradiated to aplasia, and then cells

    15    were separated and it was shown that cells could be

    16    accumulated at the site of inflammation.  In Brecher's

    17    original studies, he showed that some cells could get

    18    there and they could circulate and that some effects

    19    could be seen.

    20                What happened historically from that point

    21    was really very gradual.  But a key event again here

    22    in the early 1960's was the development of the concept

    23    of transfusing cells from CML donors.  Studies that

    24    were, I would say, led by Jay Freireich but involved

    25    a number of people here and subsequently elsewhere,

                                                                         16

     1    showed that you could take CML cells, donors were

     2    patients who were untreated and recipients were

     3    patients with leukemia usually, and you could get the

     4    CML cells to circulate in those persons and the more

     5    mature of the CML cells would actually migrate to a

     6    site of inflammation.  More dramatically in those

     7    early studies it was shown that using a very simple

     8    index that the patients became afebrile.  Dr.

     9    Freireich often talked about the fact that patients

    10    treated with CML cells showed clinical evidence of

    11    improvement very rapidly.  Those studies were greeted

    12    with great enthusiasm.  I would say the enthusiasm was

    13    tempered in time as treatments for CML improved, and

    14    also it was recognized that you could have the CML

    15    cells engraft, you could transfer infection with CML

    16    cells, and that there were a variety of complications

    17    that were associated with this approach to therapy.

    18                Actually, the next period of development

    19    centered on the development of the cell separator,

    20    work that was supported and performed here by a series

    21    of investigators. I would say Dr. Seymour Perry, who

    22    many of you may remember worked here for many years

    23    studying granulocyte and leukocyte kinetics, was the

    24    real father of the NIH efforts in this regard.  But

    25    there were a series of investigations performed here

                                                                         17

     1    by Dean Buchner, Bob Epstein, Bob Graul, and then

     2    myself over a period of years which really tried to

     3    lay a physiological framework for the advancement of

     4    neutrophil transfusion.  This is a picture from one of

     5    the first papers about this topic of the centrifuge

     6    for collecting cells.  And we all know that have been

     7    involved in blood banking how centrally important the

     8    development of this centrifuge concept for separating

     9    cells on the basis of their density has been.  So it

    10    was great research done by the IBM in collaboration

    11    with the NIH, and it did lead to the capacity to

    12    procure large amounts of cells.  

    13                This is a very brief slide about Dean

    14    Buchner's work, studies which were originally done in

    15    dogs, and showed that you could collect of the order

    16    of 24 billion white cells if you kept a dog on this

    17    centrifuge long enough, and that you could then

    18    actually transfuse these cells and see them circulate,

    19    sort of reproducing the work that George Brecher had

    20    done, but showing it with larger numbers of cells in

    21    the same species, but showing that you could, in fact,

    22    get very good increments if you used enough cells. 

    23    And importantly, they showed in this very early study

    24    that cells collected with a centrifuge would

    25    circulate.  And subsequent to this work, Bob Epstein,

                                                                         18

     1    who worked in Seattle with Reg Clift and Don Thomas

     2    went on to show that you could use this same model and

     3    then in the irradiated dog injected with E.coli to

     4    develop bacteremia, you could in fact show an effect

     5    on an experimental infection in terms of the clearance

     6    of the infection.

     7                Now when I joined and began working with

     8    Bob Graul, he was then actually in that era just

     9    beginning clinical trials here of the use of

    10    neutrophil transfusions for patients with sepsis. 

    11    These were pioneering studies, studies that were done

    12    not in a rigorous controlled trial but studies

    13    published in the New England Journal, which clearly

    14    suggested that this was a promising area for

    15    application.  The original studies were done primarily

    16    but not exclusively with centrifuge collected cells. 

    17                The studies that I was involved in here

    18    with Bob and Herb Reynolds and a number of other

    19    investigators involved dogs, again irradiated to

    20    produce neutropenia, and then injecting those dogs

    21    with Pseudomonas aeruginosa intratracheally to cause

    22    a localized pneumonia, and then treating the dogs in

    23    a randomized controlled rigidly monitored study where

    24    some dogs were supported with platelets only and the

    25    others were supported with platelets with

                                                                         19

     1    granulocytes.  

     2                Just to make it a little more interesting

     3    and colorful, I can comment very briefly upon things

     4    I remember about the trial.  As I was walking around

     5    here yesterday, I was remembering I used to keep an

     6    old bicycle outside the cafeteria downstairs to ride

     7    back and forth to the animal quarters to administer

     8    all the treatments to these dogs.  And a key reason

     9    for our success in this trial is that the NIH then had

    10    a farm in Poolesville.  I think it is all developed

    11    now, but we had a farm out there with great big

    12    foxhounds who were the donors, and in our clinical

    13    trial or our preclinical trial, we used very small

    14    beagles as the recipients.  So that allowed us the

    15    advantage of despite the number of cells we could

    16    collect, we could see and measure and do a lot about

    17    neutrophil increments. 

    18                This is the picture of the lung of a dog

    19    injected unilaterally with pseudomonas and developing

    20    a characteristic hemorrhagic pneumonia in an animal

    21    with a very low neutrophil count.  The key observation

    22    we reported in these studies in the Journal of

    23    Clinical Investigation in 1974 is in fact that if you

    24    looked at controls versus transfused animals, you

    25    could clear the Pseudomonas of the specific type we

                                                                         20

     1    had injected from the lung by a series of

     2    transfusions.  And we showed in our randomized trial

     3    that you could improve survival.  A number of other

     4    things came from the study, but it was the place that

     5    I personally became convinced if you had enough cells

     6    that were functionally intact that you could use this

     7    approach to treating severe infections.

     8                We also studied at this period the

     9    interaction of antibiotics and neutrophils and derived

    10    certain conclusions about that, and I think that is

    11    another important consideration that will come up when

    12    any clinical trial is now considered, that is, which

    13    antibiotics are best and how to approach the

    14    antibiotic neutrophil interaction.  

    15                In terms of what happened then is a nice

    16    illustration, I think, of the circuitous path of

    17    clinical research.  This is a picture of a 1970's

    18    filter that we used and was used widely then to

    19    collect neutrophils by filtration leukopheresis.  Many

    20    of you will remember this.  It basically depends upon

    21    the property of neutrophils to stick to anything

    22    almost, and that is in fact the way that they are

    23    selectively recruited to a site of inflammation, and

    24    this basic stickiness of the cells was how they could

    25    be collected in much larger numbers.  Many of the

                                                                         21

     1    studies performed in the 1970's involved the use of

     2    this filtration system as a way of trying to increase

     3    cell numbers.  

     4                And what happened is that in the

     5    development, it was learned that you could improve

     6    collections if you used starch to accelerate red cell

     7    sedimentation and if you used steroids to raise the

     8    counts in the blood.  But what really helped in terms

     9    of the numbers was the use of a filter to get lots of

    10    cells.  This is data from that era showing a

    11    comparison of how many cells you could obtain at best

    12    with a centrifuge and how many more you could collect

    13    by filtration leukopheresis.  These are probably

    14    conservative differences.  That is to say if it were

    15    three to five times as many that that would be the

    16    expected.  The problems that occurred were that

    17    although the efficiency of collection was more, the

    18    cells were damaged in the process of their collection. 

    19    And in fact probably in the process of activating

    20    cells, we would say in modern terms the release of

    21    cytokines from neutrophils, you often saw febrile

    22    transfusion reactions in response to the

    23    administration of filter adherence collected cells.

    24    Nevertheless, until around 1980, this technique was

    25    widely used.  

                                                                         22

     1                There were a number of studies in this era

     2    that sort of took on, if you will, the filtration

     3    leukopheresis cells and looked at what was wrong with

     4    them.  This is a paper by Tom Price, who is speaking

     5    next, about this, looking at the difference in the

     6    disappearance rates of various types of neutrophils,

     7    cells from phlebotomy, cells collected by

     8    centrifugation, intermittent flow centrifugation, and

     9    then cells collected by filtration leukopheresis.  And

    10    basically the cells were -- it was found that the

    11    cells were damaged enough in the collection process

    12    that they wouldn't circulate.  And although there was

    13    suggestive evidence that they might be useful for

    14    therapy, the evidence was never very strong.  And

    15    particularly because of the transfusion reactions,

    16    this process of collecting cells by filtration

    17    leukopheresis fell out of vogue.  

    18                Now there have been many summaries of the

    19    studies that were done in the 1970's and early 1980's

    20    looking at the benefit and the use of granulocytes

    21    based on various trials.  This is a slide borrowed

    22    from the summary work by Ron Strauss outlining what he

    23    would consider the best of these trials, dating from

    24    Bob Graul's trial that I mention here as the first of

    25    these, up until a trial performed at UCLA in 1982.  So

                                                                         23

     1    it spans a 10-year period.  Those of you interested in

     2    clinical trials will say aren't these pretty small

     3    numbers, and they really are.  In fact, the number of

     4    patients in the treated groups are sufficiently small

     5    that part of the problem with these trials was just

     6    basically their size.  Another problem is that cells

     7    in these trials were collected by various techniques,

     8    and in general the trials that showed the best results

     9    involved the transfusion of the largest numbers of

    10    cells.  

    11                Another of the problems in these trials is

    12    that the patients weren't always the same.  That is

    13    reflected probably most easily here if you look at the

    14    percent survival of the control groups.  Because if

    15    the comparison group did well, it was very difficult

    16    to imagine that you would show a benefit of the

    17    treatment.  So, for instance, in a study like the last

    18    study with a 72 percent survival rate in the control

    19    groups, the fact that the transfused group did more

    20    poorly, these numbers are probably not different, but

    21    this is so high it is hard to imagine that this trial

    22    would have shown a benefit.  Suffice it to say the

    23    clinical trials were not sufficiently convincing that

    24    although there were people who spoke enthusiastically

    25    about this topic for a number of years, clinicians in

                                                                         24

     1    general dropped this idea because of the results of

     2    these randomized trials and the difficulty in seeing

     3    the benefit to a patient of a single transfusion.

     4                If you look in more detail at these trials

     5    though by specific types of infection, you can see

     6    that for certain kinds of infections it appears that

     7    patients really did do better.  And I would say that

     8    Ron Strauss, who wrote this report, were he here would

     9    say that he believes that what these trials showed is

    10    a benefit, but that the trials were not sufficiently

    11    convincing to convince everyone of that.  

    12                Probably the most positive and most recent

    13    trial of note is the trial that was performed by Mitch

    14    Cairo and associates, a trial that involved an

    15    interesting comparison.  These were in children, so

    16    you had the advantage of small recipient/big donors,

    17    but you also had a comparison group, that is, the

    18    comparison of neutrophil transfusion versus a control

    19    which involved gamma globulin injections in neonates. 

    20    What this trial showed is that there was a very

    21    significant benefit in neonates, but critics of the

    22    trial have said that the two groups in the study were

    23    not really comparable and that the methods of

    24    randomization lacked the rigor to make this study

    25    really a definitive study for the treatment of

                                                                         25

     1    neonates with sepsis, and in fact it is for this

     2    reason probably that this trial was never really

     3    accepted as convincing evidence or the practice of

     4    neutrophil transfusion in neonates, despite these very

     5    striking results, has never been widely introduced.

     6                Now paralleling these efforts by many

     7    people to develop neutrophil transfusion was another

     8    development that is very important for the reason that

     9    we are here today.  And that is the development of our

    10    understanding of the regulation of granulocyte

    11    production and the use of the colony stimulating

    12    factors in patients and in normal subjects to try to

    13    raise the neutrophil count.  And because it is so

    14    basic to our discussions, I thought I would review

    15    this background information with you as well as a part

    16    of my history talk.

    17                Many of you will recall that in the mid-

    18    1960's, it was learned that you could take bone marrow

    19    cells in a petri dish and with a tissue culture media

    20    and some source for the stimulating factor, the cells

    21    would grow and form colonies.  Again reflecting back,

    22    it was during my years here that this technique came

    23    along, and it was a very exciting development with

    24    Paul Carbone and Clarence Brown.  We did the original

    25    colony assays here at the NIH showing that you could

                                                                         26

     1    grow cells because this led to so many important

     2    developments in the whole field of hematology,

     3    oncology, and transfusion medicine.  

     4                Suffice it to say the field has come a

     5    long way.  And I would like to make just a couple of

     6    important points related to this slide about the

     7    various growth factors involved in regulating

     8    leukocyte production.  The key concept is that early

     9    cells have lots of surface receptors affecting the

    10    formation of cells, but the late stage of development,

    11    or more specifically, the regulation of the number of

    12    circulating neutrophils is governed by a single

    13    factor, G-CSF.  That is to say that an animal made

    14    deficient in G-CSF does not have a normal circulating

    15    count.  And although they make neutrophil precursors,

    16    they don't mount a normal neutrophil response usually

    17    or in response to infection.  So the G-CSF, one of the

    18    reasons that we are talking about it today is that it

    19    is the natural regulator of the level of circulating

    20    neutrophils in the same way that erythropoietin is the

    21    natural regulator of the circulating level of red

    22    cells and thrombopoietin is the natural regulator of

    23    the level of circulating platelets.

    24                Now G-CSF as a drug was introduced in the

    25    late 1980's, and many of you know a great deal about

                                                                         27

     1    it.  The gene was originally isolated from a bladder

     2    cell line, a cell line that was from a patient with

     3    neutrophilia.  In fact, many malignant cells

     4    overproduce G-CSF and served as the original source

     5    for the material that was used in developing the basic

     6    structure of this protein and understanding its

     7    genetic regulation.  

     8                The pharmacological effects of injecting

     9    G-CSF are now well characterized and most of you know

    10    then.  That is, if you inject this drug, you can

    11    quickly achieve levels that are far higher than you

    12    normally achieve with infection or stress or a variety

    13    of other natural stimuli.  The drug is prepared and is

    14    easily administered and in fact has relatively few

    15    side effects.  This will come out further as the

    16    conference proceeds.

    17                In terms of how the colony stimulating

    18    factors work, just a few key points.  One is we heard

    19    a lot yesterday about the use of G-CSF, and you could

    20    say that for GM-CSF too.  That is, they are agents

    21    which mobilize the earliest hematopoietic cells from

    22    the marrow to the blood.  The details of exactly how

    23    that works are still not know.  But it is a dramatic

    24    effect, an effect that was totally unexpected when

    25    these agents were originally studied.

                                                                         28

     1                G-CSF is particularly powerful also to

     2    stimulate the flow of cells down the pathway or as I

     3    usually describe it, to squeeze down the production

     4    time for neutrophils from early cells to mature cells

     5    in the marrow.  So like an accordion, you squeeze down

     6    this time dimension and you get more cells and you get

     7    them quicker.  And finally, because you have more

     8    cells -- well, G-CSF also releases the mature cells

     9    from the marrow to the blood and then finally because

    10    you have more cells in the circulation, it allows for

    11    the possibility of a larger inflammatory response.  So

    12    you can see that this natural stimulus, that is, it

    13    arises in infection, or as the drug might be used to

    14    stimulate neutrophil production has a multiplicity of

    15    effects.  And you can imagine that in the development

    16    of this agent that there have been many potential

    17    clinical applications.  We are talking today just

    18    about one of them.

    19                We began in Seattle to try to investigate

    20    and to build this picture further, now about 8 years

    21    ago, about 1990.  Our original studies were a trial

    22    that we did to try to compare the effects of G-CSF in

    23    young and elderly subjects.  Our original idea was to

    24    try to study aging.  That is, we wondered if there was

    25    an impairment of the proliferative capacity of cells

                                                                         29

     1    as people get older.  So we compared healthy young

     2    people and healthy elderly people and we had a regimen

     3    which involved G-CSF daily for two weeks and then a

     4    whole variety of measurements.  I am not going to

     5    dwell upon many of these, but just to show you some

     6    highlights from studies that we have done then over

     7    the last eight years.

     8                This was the original curve showing that

     9    normal people have a very stable neutrophil count with

    10    no injection.  If you give a small dose of this drug,

    11    you get a small effect.  If you give a larger dose --

    12    this is 30 mcg total dose per day and this is 300 mcg

    13    total dose per day injected once in the morning

    14    subcutaneously measuring morning counts, and what you

    15    see are these characteristic patterns.  And if we had

    16    gone up higher in the dose, there probably is a

    17    plateau, but no one has ever really measured how high

    18    that plateau may be in terms of driving neutrophil

    19    production with this drug.  But you can see with a

    20    dose of 300 mcg daily, you can get to a plateau count

    21    of roughly 25,000 in healthy people fairly quickly. 

    22    And in fact it is this rapid increase which

    23    distinguishes the effects of G-CSF from GM-CSF when

    24    administered to normal subjects.  GM-CSF causes a much

    25    more gradual rise in the count over a more protracted

                                                                         30

     1    period.

     2                If you look at the cells that are produced

     3    in this kind of a setting, show on the left-hand side

     4    are normal neutrophils and the same person's blood

     5    looked at 5 days later after daily administration of

     6    G-CSF.  It is a gradual transition.  But what you see

     7    is the production of bigger, bluer cells with a

     8    somewhat less mature nucleus.  And if you look in

     9    greater detail, you can see a number of other

    10    interesting features of the cells.  These are

    11    sectioned electron micrographs which show normal

    12    neutrophils and cells from a person treated for five

    13    days with G-CSF.  And as you can easily see, the cells

    14    are bigger.  If you look more carefully at the cells,

    15    you can see the surface of the cells are smoother. 

    16    You can see in these cells bits of endoplasmic

    17    reticulum or what would be referred to in a laboratory

    18    as delivery bodies.  You can see differences in a

    19    variety of things, including probably the average size

    20    of the granules.  Suffice it to say the stimulus

    21    changes many aspects of the cell morphology, but in

    22    general produces cells that are younger looking.

    23                If you look by scanning electron

    24    microscopy what you see are if this is a normal

    25    neutrophil with its rugged surface -- this is a

                                                                         31

     1    neutrophil from someone treated for 5 days with G-CSF. 

     2    There is more redundancy to the membrane of the cell,

     3    and you can see lots of these cells that look like

     4    this with scanning electron microscopy.  And it is a

     5    reflection of the fact that as neutrophils mature,

     6    like as people mature in general, they shrink and the

     7    membrane shrinks around them.  So these changes are

     8    not totally unpredictable in terms of general cell

     9    biology, but they are rather dramatic to look at.  I

    10    have often described these cells as looking like

    11    someone running down the hall with their white coat

    12    flapping behind them.  And there are probably many

    13    features of how the cell functions that are slightly

    14    different for these cells versus these cells, but in

    15    general the cells have the same basic function. 

    16                Tom Price and I did studies in this era of

    17    investigation looking at how much does G-CSF stimulate

    18    the flow of cells from the marrow to the blood.  And

    19    this is kind of a classic study, something I learned

    20    to do here from Seymour Perry.  Studies which show if

    21    you injected tritiated thymidine and look at the

    22    yellow curve here, this is the normal emergence time

    23    for a neutrophil from the marrow.  That is, you label

    24    with tritiated thymidine the cells at the last stage

    25    of cell division, and then watch for those cells to

                                                                         32

     1    appear in the blood.  There is normally a lag time of

     2    about six days.  This has been known since the 1950's. 

     3    If you inject G-CSF, what you can show with our

     4    studies is that you shift this curve to the left.  The

     5    30 mcg curve shifted it this much and the 300 mcg

     6    curve shifted it this much.  That is, you reduce the

     7    post mitotic transit time for the neutrophil from

     8    roughly 6 days to 3 days or reduce it by 50 percent. 

     9    That is a big change, and so much of what you see

    10    reflects this pushing of the cells down the pathway

    11    and getting them into the blood sooner, younger, and

    12    looking as I just showed you.

    13                If you look at a schematic of what happens

    14    when you give G-CSF to a normal person for a period of

    15    time, you go from a schematic that looks like this

    16    with each of these bars representing a cell between

    17    divisions, and the number of divisions reflected by

    18    the number of forks along the road.  You can see that

    19    if you give G-CSF, either as a drug or if people

    20    produce it in response to infection, you amplify the

    21    number of cells produced, and you do it in a shorter

    22    period of time by reducing primarily the G-zero phase

    23    of cell development.

    24                If you look at the function of these cells

    25    -- we have done a number of studies and this is

                                                                         33

     1    terribly relevant to the development of this field. 

     2    If you look at neutrophil function after

     3    administration of G-CSF, what you see depends upon

     4    when you look.  Because you are looking at a dynamic

     5    circumstance.  Now this is a graph from work we

     6    published a few years ago, work with Bob Allen.  Other

     7    people have done this general line of work.  But if

     8    you look at the three groups of subjects from this

     9    trial we did -- control, 30, and 300 mcg treatment --

    10    and if you use a low stimulus like PMA in a low

    11    concentration, what you observe is that cells from the

    12    blood of a person treated with G-CSF are primed but

    13    they are not actually stimulated or activated by the

    14    treatment.  And that is to say if you take the cells

    15    from the blood and you expose them to a low dose or a

    16    low amount of this stimulus, you really see no effect

    17    of treatment.  However, if you use a high dose of PMA

    18    or some other agonist, what you can show is that there

    19    is a time-dependent change in the response of the

    20    cells to the stimulus.  Now these colored bars at the

    21    bottom are just a reproduction of the data I showed

    22    you a moment ago about emergence time.  The purple is

    23    the shortest emergence time, which refers to the

    24    highest peak here in terms of a change in the PMA

    25    response of the cells.  And that is to say if you give

                                                                         34

     1    a higher dose of G-CSF, you get cells produced in the

     2    blood which are, again, not activated, but they are

     3    primed to a greater degree to make a bigger response

     4    to an agonist like PMA.  

     5                Now why does this happen?  I think a basic

     6    underlying concept is that if you administer a

     7    powerful cytokine like G-CSF, you actually alter many

     8    aspects of the cells.  And we believe this occurs

     9    because of effects on coordinated gene expression. 

    10    Not only are you inducing cell division, but you are

    11    actually inducing the enzymes that are packaged in the

    12    granules to be different than they would be normally. 

    13    And we believe that this reflects, in fact, a

    14    plasticity in the production of neutrophils that

    15    occurs with infections and that is simulated by growth

    16    factor administration, so that the effects of

    17    treatment, like infection, are actually to produce

    18    cells that are more effective than normal cells would

    19    be in adaptation of the host, as we have learned over

    20    many years occurs in tuberculosis and in other kinds

    21    of infectious diseases.

    22                Now if you look at the cell surface.  I

    23    showed you pictures -- if you look at the cell surface

    24    and look at the various properties of cells, a number

    25    of investigators have shown this.  What you see,

                                                                         35

     1    again, in terms of surface properties of neutrophils

     2    depends on when you look.  If you look at one day of

     3    G-CSF treatment, actually the circumstance for most

     4    treatment and then transfusion studies, you find

     5    relatively little change.  If you look at longer

     6    periods of time, though, you can see effects on

     7    various markers for neutrophil adherence and for

     8    function.  And shown down here, for instance, in this

     9    corner is the substantial enhancement of the

    10    expression of CD14, a binding moiety for endotoxin

    11    that is induced on neutrophils by G-CSF treatment. 

    12    Probably greater than any of these is the effect on

    13    the expression of the high affinity receptor for IgG

    14    on neutrophils, which is greatly induced by G-CSF

    15    treatment.  And it is conceptually important in terms

    16    of the internalization of bacteria by neutrophils and

    17    their killing of the organisms.  But the full benefit

    18    of that effect is not really known.

    19                In terms of some effects of these changes,

    20    though, there is an interesting experiment that my

    21    colleague Conrad Liles did and published just last

    22    year.  This is looking at the killing of fungal

    23    organisms, a focus of interest in neutrophil

    24    transfusion therapy, and looking at the effects of G-

    25    CSF treatment of a normal person or potentially a

                                                                         36

     1    normal donor and looking at the capacity of the cells

     2    to kill this class of organisms.  And what this shows

     3    is the purple bars being the controls and neutrophil

     4    killing then for three classes of organisms --

     5    Candida, Aspergillus, and Rhizopus.  And what this

     6    shows is if you look at neutrophils from a normal

     7    person after five days of G-CSF, there is really no

     8    effect on the killing of Candida, which are relatively

     9    easily killed by a neutrophil.  However, for

    10    Aspergillus and Rhizopus, you can show in this kind of

    11    a model the induction of an enhanced capacity to kill

    12    these organisms.  These experiments were done with

    13    spores.  We are currently doing experiments in Seattle

    14    now looking at the hyphae forms of these organisms. 

    15    But suffice it to say that there is considerable

    16    evidence to say that you can use cytokines not only to

    17    enhance the number of cells the body produces but also

    18    the functional capacities for critical functions like

    19    this of these cells.

    20                This is sort of a summary of what I have

    21    told you.  G-CSF in this setting and what is relevant

    22    is it increases production by accelerating release of

    23    cells leading to the shift of band neutrophils into

    24    the blood and other slightly immature cells.  The

    25    cells that are released are primed for an enhanced

                                                                         37

     1    metabolic response.  If you use the right organisms,

     2    you can show that you have enhanced microbicidal

     3    activity, and actually there are a number of other

     4    changes that occur, most of which are changes which

     5    would enhance the body's response in an infection.

     6                Now one of the most interesting effects of

     7    cytokines on neutrophil production that is important

     8    in the development of our concepts today is the effect

     9    on cell viability.  This is a panel from work that

    10    Conrad Liles did a few years ago actually looking at

    11    just taking a test tube of blood, if you will, or

    12    isolated neutrophils and looking at how long those

    13    cells survive in vitro.  What this shows is normally

    14    the blue line neutrophils poop out, right?  You know

    15    that.  If you leave a tube of blood in your pocket and

    16    forget to do a count today and test it tomorrow, the

    17    count is lower.  Neutrophils die by the  process of

    18    apoposis, their natural process of death, and you can

    19    show this in the laboratory very nicely that they fall

    20    off over time.  This has been a central issue in the

    21    conceptualization of how you would ever supply

    22    neutrophils for transfusion therapy because they don't

    23    last very long.  Well, what has been shown by a number

    24    of investigators now is addition of G-CSF and GM-CSF

    25    or interferon gamma, these are all agents which tend

                                                                         38

     1    to prolong the in vitro survival of neutrophils. And

     2    as I will show you in a moment, they also promote the

     3    in vivo survival of these cells.

     4                Now we took these ideas first to the

     5    clinic in about 1993 in work that was performed at the

     6    Puget Sound Blood Center in Seattle and at the

     7    Hutchinson Cancer Center, and actually there were a

     8    number of other groups around the world who were

     9    interested at the same time -- a group of

    10    investigators here and in several other centers

    11    particularly, including the M.D. Anderson Hospital in

    12    House.  The basic idea that we investigated in Seattle

    13    was the concept of providing neutrophil support for a

    14    person after bone marrow transplantation to keep their

    15    counts from going low.  And because we were concerned

    16    about alloimmunization and other problems, we used the

    17    actual marrow donor, an unfortunate circumstance where

    18    we had some twins and syngeneic individuals, so that

    19    we could try to optimize neutrophil support through a

    20    period of neutropenia using cells collected from G-CSF

    21    stimulated donors. 

    22                This is a summary of the basic plan.  We

    23    had seven people.  We used G-CSF at 5 mcg per kilogram

    24    per day.  The people donated an average of, as you can

    25    see, just over 7 times.  We collected cells by

                                                                         39

     1    centrifugation and used starch to accelerate red cell

     2    sedimentation.  And because it was being done

     3    repeatedly, these volunteers became patients in a

     4    sense as they had a subclavian catheter implanted. 

     5    The controls were historical controls of other people

     6    not given G-CSF.  These were the rather dramatic

     7    results of this trial, which was published in Blood in

     8    1993.  Bill Bensinger is the senior author.

     9                What the trial showed is compared to no G-

    10    CSF, that the number of cells that were collected were

    11    roughly tenfold higher.  And more importantly, the

    12    increments in the blood of the recipients were almost

    13    tenfold higher, with counts measured 24 hours after

    14    the transfusion.  Now if you are familiar with this

    15    field, you know that for many years, you could

    16    transfuse lots of cells but you couldn't count them. 

    17    In fact, it was interesting to review some old papers. 

    18    If you look at determining hematopoietic recovery

    19    after transplantation even though you are giving

    20    granulocytes, it wasn't difficult in the old days

    21    because you could transfuse the cells.  There were

    22    never any in the circulation, so you could still see

    23    when the marrow recovered.

    24                Actually in this trial and in I would say

    25    other work since then, what you find is you get enough

                                                                         40

     1    of an increment with transfusing cells from a G-CSF

     2    stimulated donor that it makes it hard to recognize

     3    when recovery occurs.  As you can see here, we found

     4    in this trial transfusing roughly 40 billion

     5    neutrophils a day that we got a median increment at 24

     6    hours approximately of 570, and a mean increment of

     7    nearly 1,000.

     8                Now we weren't satisfied.  And actually

     9    the following summer, using a medical student for a

    10    graduate honors project, we conducted a randomized

    11    trial of giving G-CSF with and with dexamethasone to

    12    see if we could use these two agents together to get

    13    the counts even higher.  Now being interested in this

    14    field for a long time, I was skeptical that it would

    15    make any difference, but I thought it was worth a try. 

    16    This is the schedule we used, chosen somewhat

    17    arbitrarily.  We used the dose of G-CSF 300 mcg that

    18    we had used before or twice this amount with and

    19    without 8 mg of dexamethasone.  This was administered

    20    subcutaneously and this orally simultaneously, and all

    21    we did was to do blood counts over the next 24 hours. 

    22    But what you can see is if you look at the 12-hour

    23    points, that the addition of dexamethasone to G-CSF

    24    substantially increased the levels of the counts.  Or

    25    as you can see also quite easily, we went in normal

                                                                         41

     1    people from a count of 4,000 to 40,000 in 12 hours and

     2    they barely knew it.  Most of what they noticed was

     3    the effects of the dexamethasone.

     4                So we were very impressed at how much we

     5    could raise the counts.  It is still not known why

     6    this occurs.  My bet -- but there is on data to prove

     7    it -- is that the steroids actually effect the

     8    capacity of the cells to be mobilized with the G-CSF. 

     9    Probably some effect on receptor or post-receptor

    10    mechanisms of the cells.  Suffice it to say it is a

    11    big effect and again it has potentially a large effect

    12    in planning or conducting a clinical trial.

    13                What we did was to go on and conduct some

    14    studies using this combination of drugs, collecting

    15    cells, and making measurements.  This work is sort of

    16    barely history, but it is published in August of this

    17    year in The Journal of Transfusion.  Here are a

    18    picture of the cells collected in this way, nice-

    19    looking but young-looking cells.  This is just giving

    20    G-CSF and dexamethasone and collecting cells 12 hours

    21    later.  And Dr. Price in a moment will talk more about

    22    this.  

    23                If you look at the general effects on

    24    these cells, and Dr. Liles this afternoon will

    25    describe this in greater detail, but you can collect

                                                                         42

     1    75 to 100 billions cells now with this technique,

     2    which have normal functions, slight changes in their

     3    immunophenotype, and again, as I mentioned, an effect

     4    on the survival of the cells.  What we have shown with

     5    isotope labeling studies is in fact that going from a

     6    normal half-life of around 8 to 10 hours for

     7    neutrophils, the neutrophils collected in this fashion

     8    have a blood half-life of roughly 20 hours.  So they

     9    have a long survival, as I showed you in vitro, and

    10    the calculated production rates or turnover rates

    11    would be, of course, very large for large increases in

    12    the cells with a long survival in the blood.  

    13                Now just as a transition to what I am

    14    going to say about conducting a clinical trial, it is

    15    very important to know that there is enough data now

    16    to say what happens if you transfuse cells like this

    17    into patients.  We have done some studies in Seattle

    18    transfusing cells from people treated with G-CSF and

    19    dexamethasone to patients who are marrow transplant

    20    patients with serious bacterial and fungal infections. 

    21    This is just to highlight this information, but what

    22    it shows is if you transfuse a person with almost no

    23    neutrophils, you can bring their count to near normal

    24    with a transfusion and you can then go up another

    25    notch if you give a second transfusion the next day. 

                                                                         43

     1    That is, you can normalize the neutrophil level of a

     2    person with no neutrophils, something that heretofore

     3    was never possible.  

     4                It is based upon that I feel and I think

     5    several people here feel like it is time for a real

     6    reconsideration of this idea or a consideration of a

     7    clinical trial to evaluate neutrophil transfusion

     8    again.  20 years have gone by since this was really

     9    undertaken, and there have been a lot of changes in

    10    many aspects of medicine which makes this justified. 

    11    The best choice, although challenging to do, is a

    12    randomized control trial using therapeutic transfusion

    13    rather than prophylactic transfusion.  The biggest

    14    problem with this is alloimmunization if you give

    15    cells early.  So late after transplantation when you

    16    really need them, the patient might have a smaller

    17    response.  And also logistically this is a huge

    18    undertaking.  

    19                In general, the focus of a trial should be

    20    on patients who really need it.  And those patients

    21    these days in marrow transplant centers and in most

    22    intensive cancer centers are on difficult to treat

    23    organisms, particularly yeast and molds and some

    24    resistant bacteria.  We believe in contrast to earlier

    25    eras that the best way to proceed would be with

                                                                         44

     1    cooperation, a multi-center trial.  I showed you

     2    reports of small, relatively inconclusive trials, and

     3    part of the problem was how they were conducted.  And

     4    finally, you need to do them with some standard

     5    approach to the patient care, the other aspects of

     6    patient care, in order to be certain about what you

     7    see.

     8                In terms of trial design then, the best

     9    ideas are to use people who have marrows that are

    10    expected not to recover quickly, that is, they have

    11    received aggressive chemotherapy or transplant.  We

    12    believe that this is an applicable approach to

    13    patients with neutropenia at present.  There may be

    14    other ideas, but that should be the focus.  And as I

    15    mentioned, fungal infections and people preferentially

    16    chosen to be not demonstrated to be alloimmunized

    17    before transfusion support is given based upon much

    18    evidence that you can alloimmunize somebody and not

    19    get a response.  And the basic idea in a randomized

    20    trial should compare if patients have fungal

    21    infections or bacterial infections that they receive

    22    a standard therapy, a standard therapy plus

    23    neutrophils procured in what I would say is a modern

    24    way.

    25                Now there are many issues, and what I said

                                                                         45

     1    may make it sound simple, but I assure you it is not. 

     2    That is, we are still studying issues around

     3    mobilization strategies.  How much difference does it

     4    make about which drugs and how much you give and when

     5    you give them.  There are issues still about the

     6    quality of cells.  I have said that cytokines affect

     7    cell formation and function, and that diversity of

     8    effects needs to be considered in terms of the actual

     9    trial design.  There are lots of issues related to

    10    donor willingness and safety.  It is amazing in this

    11    country the diversity in terms of the willingness of

    12    people to give blood.  Reasons that are very complex. 

    13    And if you think about another layer of complexity,

    14    that is accepting the idea of being treated with a

    15    drug before you give blood, you can imagine that there

    16    are many aspects of this to be considered if a trial

    17    is to be conducted well and conducted safely.  There

    18    are many issues, some of which will come out today,

    19    about recipient benefits and risk, and then there is

    20    the question of having in a trial design good, clear,

    21    acceptable evidence of therapeutic efficacy.  

    22                In terms of how this should be done, I

    23    believe and I think many do that neutrophils are there

    24    to kill microbes.  And so a trial should be designed

    25    primarily to show an effect on microbes, that is, the

                                                                         46

     1    clearance of infection.  There are many secondary

     2    endpoints, though, that are important.  Important

     3    socially, important for patient well-being, important

     4    for the people who pay for medicine in this country. 

     5    So that one has to decide a trial with enough other

     6    information in it so that you can apply the results of

     7    a trial in the clinical and economic settings where we

     8    practice.

     9                My final point in terms of general

    10    comments about a clinical trial comparing the past

    11    with the present is that we live in a very dynamic

    12    world. Look at the paper today, right?  And in this

    13    particular field, we live in a world that is changing

    14    rapidly.  I have said enough, I bet, to convince some

    15    of you, if you weren't already convinced, if you went

    16    home tonight and it was your mother or father or

    17    sister or brother and I offered you this, you would

    18    say, of course.  And if I said you might not get it in

    19    a randomized trial, you would say, oh no, I don't want

    20    to participate.  Because times have changed.  This

    21    approach does show considerable promise.  And I think

    22    that the window of opportunity in clinical trials in

    23    this field, like in other fields, is relatively small. 

    24    Because knowing as I do people around the world who

    25    work in this particular area, many people are taking

                                                                         47

     1    this approach and taking it without really firm

     2    evidence that it is a clinical benefit.  So the time

     3    for a clinical trial is relatively brief.

     4                I am going to stop at that point.  Again,

     5    it is great to be back at the NIH and thank you very

     6    much.

     7                CHAIRPERSON HARVATH:  That was really

     8    great.  Thank you very much.  It is a pleasure to

     9    introduce the next speaker, Dr. Thomas Price, who is

    10    also a Professor of Medicine at the University of

    11    Washington and the Director of Puget Sound Blood

    12    Center.  He is going to speak to us about his

    13    experience of cytokine administration to normal

    14    granulocyte donors and some other really great

    15    information I think you are going to add as well. 

    16    Thank you.

    17                DR. PRICE:  Thanks, Liana.  Thanks to you

    18    and the organizers for inviting me here.  If I could

    19    have the first slide.  What I am going to do today is

    20    to share with you our experience, which is an ongoing

    21    experience, with a trial of neutrophil transfusion

    22    that we are doing in collaboration with the people at

    23    the Fred Hutchinson Cancer Research Center involving

    24    patients that are undergoing bone marrow

    25    transplantation.  And I would just like to tell you

                                                                         48

     1    kind of where we are with this.

     2                This slide you have seen before.  It is to

     3    kind of remind you of the mobilization that we are

     4    talking about here.  And as David said, we looked at

     5    these five different mobilization regimens here.  The

     6    dotted lines are the ones that included dexamethasone

     7    in addition to the G-CSF, and as might be obvious from

     8    looking at this slide, in the trial that I am about to

     9    show you, we picked the one that we thought was going

    10    to give us the most granulocytes.  So that is why the

    11    study that you will see and that I am going to talk

    12    about now involves the 600 mcg dose of G-CSF and also

    13    dexamethasone.  Now whether it will turn out that

    14    there is really that much difference between these top

    15    two, we won't be able to say as a result of what I am

    16    going to tell you.

    17                The other thing to note, of course, is

    18    that the timing in here is to suggest that doing this

    19    12 hours before you collect the neutrophils would

    20    probably be the best time to do this. So this is what

    21    we aimed for, but keeping in mind with the logistics

    22    of when donors can actually show up and when you can

    23    actually collect the cells. As you see, we don't

    24    always hit the 12 hours right on the mark.  

    25                The basic design of this ongoing study is

                                                                         49

     1    shown in this slide here.  This is sort of a Phase

     2    I/II study, as I said a collaborative study between

     3    the Blood Center -- this is the Puget Sound Blood

     4    Center, it is not a misprint for peripheral blood stem

     5    cells here -- and the Hutchinson Cancer Center.  One

     6    of the wrinkles on this thing is that the design here

     7    is to use community donors.  Now most of the studies

     8    that have been reported using G-CSF stimulated

     9    granulocyte donors have been when the donor has been

    10    a family member or friend of the patient, a fairly

    11    captive person that you can lasso and do this to.  The

    12    idea here was to see if we could supply granulocytes

    13    to patients as they needed them using community

    14    donors.  These are donors that are just ordinary blood

    15    donors who have volunteered to be, for the most part,

    16    platelet donors or to be apheresis donors for patients

    17    that they don't even know.  Could we involve them in

    18    such a process?

    19                As I said, the dose mobilization was to

    20    give them 600 mcg of G-CSF and 8 mg of dexamethasone. 

    21    This was done as close as possible to the 12 hours

    22    prior to the collection procedure.  The collection

    23    procedure itself was routine.  We used a COBE spectra

    24    machine.  We used the high molecular weight

    25    hydroxyethyl starch, the hetastarch, as the red cell

                                                                         50

     1    sedimenting agent, and we processed 10 liters of blood

     2    for these collections.

     3                Now the patients were all patients on the

     4    bone marrow transplant ward and the Seattle transplant

     5    ward at the Hutchinson Cancer Center.  Most of these,

     6    as you will see, were patients who had already

     7    received a transplant, although there were a few

     8    patients in there who were pre-transplant.  They are

     9    all neutropenic.  The idea was to limit this to people

    10    who had 100 neutrophils or less.  And they were people

    11    with documented fungal or resistant bacterial

    12    infections.

    13                Now the original plan or the goal of this

    14    thing were these three things listed here.  One of the

    15    things we wanted to do was to evaluate the feasibility

    16    of using community donors.  We started out by just

    17    calling some pheresis donor up and saying how about

    18    coming in and getting a shot of G-CSF.  We had no idea

    19    how easy it was going to be to convince people to do

    20    this and whether we could basically supply with any

    21    kind of regulatory these components.  We also wanted

    22    to see what we actually could get in neutrophil yields

    23    by using this sort of a mobilization strategy.  The

    24    slide before was, as David said, just some normal guys

    25    that we gave these things to and did some blood counts

                                                                         51

     1    on.  The proof of the pudding was what we would

     2    actually get when we hooked them up to a machine.  

     3                And finally, we wanted to determine what

     4    the hematologic effects were going to be in the

     5    recipients.  What was going to happen to them in terms

     6    of their counts and in terms of where these cells

     7    went.  It would have been nice to make some sort of

     8    clinical determination of whether this was efficacious

     9    in the recipients, and we were going to look at that. 

    10    But right from the beginning we knew we weren't going

    11    to have enough patients probably to really make a

    12    determination that was convincing of clinical

    13    efficacy.

    14                Let me turn a little bit to the community

    15    donor recruitment business.  The way this worked was

    16    we have got this pool of 4,000 or 5,000 people who

    17    have signed up to be pheresis donors.  For the most

    18    part, these people are platelet donors, but they are

    19    also subject to being called for a granulocyte

    20    collection which traditionally has involved taking

    21    some prednisone as a stimulating agent.  The idea was

    22    that this regular donor list was that the donor would

    23    be contacted by the regular pheresis scheduler,

    24    somebody that they would almost know because they talk

    25    with them fairly frequently, and be informed that we

                                                                         52

     1    had a study going that we thought was likely to be

     2    able to improve the product, but it did involve them

     3    taking a drug and would they be interested in

     4    participating.  If they were, then they were scheduled

     5    for a donation, but they were also then put in contact

     6    with the study nurse coordinating this study who went

     7    over the study in detail with them and basically went

     8    through the informed consent procedures as to what

     9    this was all about.

    10                Then what happens is the 12-hour ahead of

    11    time visit, the donor comes in to one of the Blood

    12    Center's fixed sites.  We have five sites scattered

    13    around the Seattle area and the donor could come to

    14    any one of these sites 12 hours before.  Now as a

    15    practical matter what this meant is that we tried to

    16    shoot for 12 hours, but as a practical matter it was

    17    somewhere between 8 and 16 hours ahead of the

    18    scheduled leukopheresis.  What this means is that you

    19    can't just do a leukopheresis at any time.  The

    20    leukopheresis had to be either scheduled at the crack

    21    of dawn so that the donor could come in the evening

    22    before at a reasonable hour -- come in at dinner time,

    23    you know 7:00 or 8:00 at night, and then we could do

    24    an early morning collection.  Or alternatively, the

    25    donor could come in at the crack of dawn for the pre-

                                                                         53

     1    visit and have the blood drawn at 7:00 in the morning

     2    and then we would be able to do a leukopheresis

     3    procedure in the 5:00 in the afternoon sort of range. 

     4    But it does sort of limit you because you can't do a

     5    collection procedure at 1:00 in the afternoon because

     6    there is no right timing for the donor to come in for

     7    the ahead of time visit.

     8                Anyway, when they do come in, the consent

     9    form is signed.  They go through a preliminary donor

    10    screening, just to make sure that there are no

    11    surprises there and 12 hours later we are not going to

    12    find out that the guy had hepatitis last year.  We

    13    also draw blood for the routine things. We draw blood

    14    for a CBC and also for the ordinary testing -- for the

    15    ABO, the Rh antibody screen and for infectious disease

    16    testing.  And then, of course, the G-CSF is

    17    administered subcutaneously and the dexamethasone is

    18    given for the donor to take.

    19                Now since this is an FDA sponsored

    20    conference, I did want to make one what I think is

    21    important point about the infectious disease testing. 

    22    What we did in this study is considered that the

    23    testing that we did on the sample obtained 12 hours

    24    prior to the leukopheresis, we considered this to be

    25    the testing of record for the collection.  Now for

                                                                         54

     1    those of you who are in blood banking, this is not the

     2    usual FDA approach to things.  The usual FDA approach

     3    is to say that you have got to draw the blood sample

     4    that you are going to do for the testing actually at

     5    the time that you are collecting the blood component. 

     6    Now I think it is important, though, that we be

     7    allowed to do this as I have described it here and

     8    count this as the testing of record because of

     9    basically what David was saying about the storage

    10    capabilities of these cells.  With the current

    11    techniques, the neutrophil integrity is likely to be

    12    compromised if we store it waiting for these tests. 

    13    With the more sophisticated testing -- I mean, stuff

    14    that we have done in the past basically has shown that

    15    if you store cells for 24 hours, in terms of the

    16    cell's ability to localize to an inflammatory site,

    17    the cell loses about 75 percent of its activity at 24

    18    hours.  So it is very important, at least now, to give

    19    these cells as soon as possible after collection and

    20    we can't really wait until all the testing is done. 

    21    And this is getting worse because the time for testing

    22    keeps getting longer.  When PCR comes along, it is

    23    even going to be worse.  Also, it is not -- you know,

    24    the testing lab is not necessarily right next door

    25    these days to the place that you are drawing this

                                                                         55

     1    blood.  It may have to be sent off to a different

     2    contract place that is doing the testing.  So this

     3    timing gets worse and these things do not store well. 

     4    So I guess I am making the pitch that should the FDA

     5    decide they want to write some rules for this, that it

     6    would be very important to be allowed to have the

     7    testing of record be this sample that we draw the day

     8    before, let's say.

     9                Now it may turn out, to be optimistic

    10    perhaps, that one of the effects of G-CSF will be that

    11    we will be able to store these cells better.  But

    12    there is really no data on that in vivo yet.  So that

    13    is just totally an unknown right now.

    14                Okay, well what happened?  We had as part

    15    of this -- what I am going to tell you about so far is

    16    our experience with 19 patients that we were trying to

    17    provide granulocyte support for.  If we started at the

    18    time that these patients were identified up until the

    19    time that granulocytes weren't needed any more, there

    20    were 233 slots that had everything gone swimmingly we

    21    would have had a collection for each of these slots. 

    22    As I said, we have about 4,500 donors in our pool that

    23    we have available to call.  One of the things that

    24    surprised us, I guess, was that when we did contact

    25    these donors and said how would you like to do

                                                                         56

     1    something kind of experimental and get a subcutaneous

     2    injection of a drug, about two-thirds of them said

     3    fine, I will be right in.  Now because of various

     4    logistic things and part of having to deal with this

     5    timing that I am telling you about and the time slots

     6    that had to be available and the donor had to be able

     7    to fit, in fact we only succeeded about 75 percent of

     8    the time in getting somebody actually when we wanted

     9    them.  That was more of a logistic problem than it was

    10    a problem of not being able to find a donor.  

    11                What I am going to report to you now is

    12    the results of 175 collections with this stimulation

    13    here, this 600 of G-CSF and 8 of dexamethasone.  A

    14    little bit about donor side effects.  You have heard

    15    many times and those of you who were here yesterday

    16    heard again the story of donor side effects from G-

    17    CSF.  The experience we have had at the Blood Center

    18    has been similar to all of this.  Most of the donors

    19    experienced some side effects from this pre-

    20    stimulation.  Mild to moderate in the vast majority of

    21    donors.  With these 175 donors, 40 percent experienced

    22    some sort of bone pain, 30 percent headache, 30

    23    percent insomnia, probably more due to the

    24    dexamethasone than to the G-CSF.  About a third of

    25    these patients or a quarter to a third of the patients

                                                                         57

     1    had no side effect at all.  

     2                Now the severity of these side effects I

     3    think you can judge by the fact that of the donors

     4    that donated, when we asked them later how big of a

     5    deal this was, 98 percent of them said that they would

     6    be more than willing to come back and do it again.  

     7                What was the experience in how much we

     8    actually got.  The donors neutrophil count at the time

     9    prior to getting the G-CSF on the sample that we drew

    10    12 hours ahead of time was normal.  It averaged 3,700

    11    with this sort of range.  The time interval between

    12    getting G-CSF and the beginning of the collection

    13    averaged 13 hours.  As you can see here, it varied

    14    with an extreme for 5 and 23 hours.  The donor

    15    neutrophil count right before the collection was

    16    almost 31,000, varying here between 14,000 and 56,000. 

    17    This is the neutrophil count now and not the white

    18    count.  And the number of neutrophils that we got

    19    averaged 82 billion.  It ranged between 24 billion and

    20    144 billion.  Now just to remind you of the numbers

    21    that David showed you before, the traditional

    22    neutrophil yield that is obtained by modern cell

    23    separators using corticosteroids alone as a stimulus

    24    is usually in the 20 to 30 billion range.  So you can

    25    see here that this now is two to three times the yield

                                                                         58

     1    that you normally get without using G-CSF.

     2                Now this slide here shows you the

     3    relationship between what the donor's neutrophil count

     4    is right before the pheresis procedure and what we get

     5    in terms of the yield going all the way up to -- I

     6    can't really read that but it looks like 160 billion

     7    cells there.  This, again, is neutrophils and not

     8    white blood cells.  I think you can see that there is

     9    a little bit of scatter here, but there is clearly a

    10    pretty linear relationship between what the donor's

    11    neutrophil count is and what you get out of this.  It

    12    nicely extrapolates down to zero.  What you see here

    13    actually as the small dots are the 175 dots

    14    representing the donors that I am telling you about. 

    15    The heavier dots there are another roughly 20

    16    collections that we have done where we used only 600

    17    mcg of G-CSF and did not give the donor dexamethasone. 

    18    I think you can see that you basically get a lower

    19    neutrophil count and you get less yield.  Remember

    20    that the average neutrophil count in the 175 was about

    21    30,000.  The average for the G-CSF alone is about

    22    22,000.  The average yield is 82 billion for the

    23    combination and about 56 billion for the G-CSF alone. 

    24    But the interesting point is that the G-CSF alone

    25    dots, the big dots, appear to be in the same continuum

                                                                         59

     1    as the other dots.  It is just a matter of how high

     2    you get the count.  So it looks like this is kind of

     3    a validation of the idea that the higher you get the

     4    count, the more cells you are going to get.  It is

     5    sort of a no-brainer.  But if we could figure out a

     6    way to routinely get the cell count up to 60,000, we

     7    might be able to get a lot more cells.  We might get

     8    the average yield up to 160 billion.

     9                Now who are the recipients here?  These

    10    are the 19 patients that we gave these cells to.  15

    11    of them had had a bone marrow transplantation and 4 of

    12    them, as I said before, were pre-transplant.  16 of

    13    these patients had a fungal infection, 8 fungemia and

    14    8 an invasive infection.  Most of these are

    15    Aspergillus infections, either pulmonary or sinus

    16    infections.  And 4 of the patients had resistant

    17    bacteremias.  You can see that this adds up to 20,

    18    which means that one of the patients had two

    19    infections.

    20                Another item which I think is important to

    21    note about these guys is that in general this

    22    population was not an alloimmunized population.  We

    23    did HLA antibody screens at the beginning of the

    24    transfusion support on all these folks and the screen

    25    was negative in 15 of these 19 patients.  There was a

                                                                         60

     1    little positivity, but not very much.  The PRAs were

     2    less than 8 percent in 4 of them.  So none of these

     3    patients was highly alloimmunized to begin with and

     4    most of them had no evidence of alloimmunization.

     5                What did we see in the patients in terms

     6    of the hematologic results?  There was an average of

     7    8.6 transfusions per patient.  It ranged from 1 to 25

     8    transfusions.  You have already seen that the average

     9    dose delivered was 82 billion cells.  Now this is what

    10    happened to the patient's neutrophil count.  I have

    11    listed two things here.  One is the one-hour

    12    increment, that is, comparing the neutrophil count one

    13    hour after the transfusion with the count immediately

    14    prior to the transfusion.  You can see here that the

    15    average was about 2,600.  And as David mentioned, this

    16    is in marked contrast to the usual experience with

    17    granulocyte transfusions where one didn't see any

    18    increment and we always used to say, well, that is

    19    because they are all doing what they are supposed to

    20    do and going to the site of infection.  But in this

    21    situation, you actually do see a substantial increment

    22    in the neutrophils.  And you can see that this varies

    23    from one guy who actually had a negative increment who

    24    had a fairly high count to begin with pre-transfusion

    25    to a very high neutrophil increment.

                                                                         61

     1                The other thing to note is that these

     2    cells stick around.  They do drop off as the day goes

     3    on, but if you do a count the next morning, the

     4    average count in these people was 2,600.  This is just

     5    a coincidence that these happen to be the same number

     6    -- varying anywhere from nothing to 15,000.  So that

     7    you can see on the average we are taking patients who

     8    begin severely neutropenic and we can, again on

     9    average, convert them from somebody severely

    10    neutropenic to somebody who has a sustained neutrophil

    11    count which is normal or near normal.  Now I will also

    12    take you back to the other slide David showed you of

    13    the sequential days and the sawtooth sort of thing

    14    where if you would actually pull out this next AM

    15    count, of course you start out at zero in these

    16    patients and after the first -- it goes up for one day

    17    and comes back down again but not quite back down to

    18    where it started from, and then on day two you get it

    19    up a little higher and you can sawtooth this thing up. 

    20    So that the general experience is after a few days,

    21    the patient is running a neutrophil count that is

    22    often in the normal range.  

    23                Now you will note some low numbers here,

    24    though.  Two of these patients got no increments.  So

    25    this is the average, but a couple of them got no

                                                                         62

     1    increment at all.

     2                Now the next thing here is what we are

     3    calling the buccal neutrophil count.  This is kind of

     4    a crude way of determining whether these cells are

     5    capable of leaving the circulation and getting to the

     6    tissue sites where it is important that they do their

     7    work.  And what we do to this thing is have the

     8    patient take 24 cc of saline into their mouth, swish

     9    it around, spit it into a can, and then by staining

    10    the cells and counting them, we can count how many

    11    neutrophils are in the guy's spit.  What we find is

    12    that when we did this before we started the

    13    transfusion support, basically this is in millions,

    14    the average is .01 with this sort of a range here. 

    15    Post-transfusion -- and generally we made these

    16    measurements the next morning after the transfusion --

    17    you can see that on average there were about a half a

    18    million cells in there with this sort of a range.  Now

    19    if we were -- just to give you an idea of the normal

    20    numbers -- if we were to do this same little test in

    21    everybody in the room here, the average amount of

    22    neutrophils in a normal person is about a half a

    23    million. So these guys on average were going from

    24    nothing up to where the amount of neutrophils in their

    25    mouthwash was normal.  So these cells are capable --

                                                                         63

     1    they don't just circulate, they are capable of

     2    extravascular migration and getting supposedly to

     3    where they are supposed to go.  

     4                Now again you can see that there is a

     5    range here.  In fact, there turned out to be a

     6    correlation between this.  The guys that got no

     7    increment in the blood also got no increment in the

     8    buccal neutrophils.  This is what you would expect, I

     9    guess, but it sort of validates that maybe we really

    10    are measuring here something that means something.

    11                What happened in terms of the side

    12    effects?  I think it has been mentioned here earlier

    13    that one of the concerns of giving much larger doses

    14    of neutrophils, particularly neutrophils that have

    15    been primed by G-CSF, the early concern was that this

    16    might give an awful -- this might sort of exaggerate

    17    transfusion reactions and might exaggerate in

    18    particular pulmonary transfusion reactions and be a

    19    dangerous thing to do.  So we are looking here then at

    20    the 175 collections, but only 165 of those ended up

    21    being transfused.  If we look at the traditional sorts

    22    of things here like chills and fever, you can see that

    23    in about 7 percent of the transfusions, one saw that

    24    patients got chills and some fever.  This meant that

    25    in these 19 patients about a third of them at one time

                                                                         64

     1    or another in one or more of their transfusions had

     2    this experience.  These were mostly mild to moderate,

     3    in fact.  And actually what tended to happen was these

     4    patients would then on subsequent transfusions be

     5    premedicated with Tylenol or something like this and

     6    most of the time they did not recur.  So they tended

     7    to be things that were easily handled by the usual

     8    pre-medications you might give.  There were an unusual

     9    number or a low number of itching-hives type

    10    reactions.

    11                Now the other thing we do since we were

    12    particularly concerned about the pulmonary reactions,

    13    is we measured oxygen saturation by oximetry prior to

    14    the transfusion and after the transfusion.  You can

    15    see here that the baseline oxygen saturation was about

    16    95 percent.  It varied between 61 and 100 percent. 

    17    Some of these patients, particularly the guys with

    18    pulmonary Aspergillus, might not start out with a

    19    normal oxygen saturation.  On average, the change was

    20    basically not existent.  But if you looked at these

    21    individual things, of these 165 transfusions, the

    22    oxygen saturation decreased by 4 percent or more in

    23    only 11 of the transfusions and by 6 or more in only

    24    three of the transfusions, and in those three it

    25    actually ended up below 90.  The important clinical

                                                                         65

     1    point to make is that in no transfusion of these 165

     2    was it ever the clinician's view that something had

     3    happened.  These were just kind of measurements that

     4    were made, but there was no obvious pulmonary

     5    deterioration that was attributed to the transfusion. 

     6    So the fear that suddenly we could have set ourselves 

     7    up for a real dangerous transfusion reaction doesn't

     8    appear to have happened.  It is important to remember,

     9    though, that these patients are not highly

    10    alloimmunized people.  It is not that kind of set of

    11    patients.  

    12                Now a little bit about the HLA

    13    compatibility.  And you can tell by the fact that it

    14    is a cheaper looking slide that this is preliminary

    15    data.  What we did is we obtained serum samples on all

    16    of these patients before we gave the first transfusion

    17    and then weekly thereafter until the patient was off-

    18    study. We also, every time a donor came in, obtained

    19    lymphocytes from that donor and froze them.  So that

    20    after we are done with the patient, we can

    21    retrospectively come back and in sort of a batch run

    22    all those serum samples against all those lymphocytes

    23    and basically do a lymphocytotoxic cross-match for

    24    each transfusion that was given.  We have those

    25    results available on 12 of the 19 patients so far. 

                                                                         66

     1    What happened was if we looked at all these serial

     2    blood samples, there appeared to be an antibody to one

     3    or more of the donors in 4 of these 19 patients.  In

     4    other words -- well, I should say 12 -- the people

     5    that are finished.  In 8 of these guys, it was clean. 

     6    The lymphocytotoxic cross-match was negative in

     7    everything.  But in 4 of them, there was a reaction to

     8    one or more of the donors, and it turned out to be 14

     9    potentially incompatible transfusions.  Now I say

    10    potentially because some of these things were

    11    situations in which a late serum sample might show a

    12    reaction to an early donor, but whether or not the

    13    antibody was there when that donor was given, that

    14    might not have happened.  But for the purposes of this

    15    analysis, I would have to assume that if it ever

    16    happened, it might have been there at the time of the

    17    transfusion.  

    18                Well, if you look at these 14 potentially

    19    incompatibles, what you find is that of the 14, none

    20    of them was associated with chills and fever in the

    21    patient.  In one of them, there was somewhat of a

    22    decrease in the oxygen saturation.  The average

    23    neutrophil increment at one hour was 2,300 and the

    24    next A.M. count was 2,200.  So that if you will

    25    remember the overall group, these were both 2,600. 

                                                                         67

     1    Just on the service of it very preliminarily, it

     2    doesn't look like these HLA antibodies that showed up

     3    during the course of this really had any influence on

     4    the transfusion reactions or the hematologic results

     5    of the transfusion.

     6                Well, how about what finally happened to

     7    the patients.  The reasons for discontinuing the

     8    transfusions are listed here.  In 7 of these 19

     9    patients, we stopped because the patient's neutrophil

    10    count was high on its own or the patient had grafted. 

    11    In 3 of them it was stopped because the infection

    12    appeared to be gone.  And in 9 of them it was stopped

    13    either in 6 because the clinical situation was

    14    determined by the clinician to be futile and support

    15    was withdrawn, and of course the ultimate futile

    16    situation when the patient died.  

    17                If you take all of these patients, 9 of

    18    the 16 survived until engraftment and 8 of the 19

    19    cleared the infection.  If we sort this out by the

    20    kinds of infection, about half of the patients with

    21    the fungal infections, whether it was either fungemia

    22    or Aspergillus, survived until engraftment.  That was

    23    true of everybody with bacteremia.  In terms of

    24    clearing the infection, about half of the fungemia

    25    patients cleared the infection, nobody with

                                                                         68

     1    Aspergillus was thought to have cleared the infection

     2    and all of the bacteremias were thought to clear the

     3    infection.

     4                Now what to make of this.  This is one of

     5    these things that you can read as the glass is half

     6    full or the glass is half empty, I think.  I would say

     7    that the general impression of the clinicians on the

     8    ward was to be impressed that this was probably useful

     9    therapy.  There were a number of these patients that

    10    anecdotally were people that they thought normally

    11    would have done very badly who ended up clearing the

    12    infection or surviving longer than they thought they

    13    otherwise would have, but that is obviously just a

    14    clinical impression anecdote style and may or may not

    15    hold up.  

    16                So in summary so far, I think we can say

    17    that perhaps surprisingly that community apheresis

    18    donors are fairly easily recruited for G-CSF

    19    stimulation, that such stimulation in normal donors

    20    results in marked neutrophilia and greatly increased

    21    neutrophil yields, that when you transfuse these

    22    concentrates into patients, this can result in normal

    23    or near normal neutrophil counts in the recipients

    24    with migration of transfused cells to extravascular

    25    sites, and that although the clinical impressions are

                                                                         69

     1    sometimes impressive and the clinicians can be

     2    convinced that they are really being useful, I think

     3    we can't really say that based on these numbers and

     4    that we really are going to need control trials to

     5    assess the clinical efficacy.  Thanks very much.

     6                CHAIRPERSON HARVATH:  Thank you very much. 

     7    That was very interesting data.  Before taking the

     8    break, Dr. Epstein, who is the Office of Blood

     9    Director in the Center for Biologics was sitting next

    10    to me and we were talking about the question that you

    11    had asked of the FDA.  So before he has to leave, I

    12    would like Dr. Epstein to address your question and

    13    then we will take our break and then we will assemble

    14    a panel after the last two speakers of the morning

    15    session.

    16                DR. EPSTEIN:  Thank you very much, Liana. 

    17    Just a brief comment.  The current regulations require

    18    the donor sample to be obtained on the day of

    19    collection, and we can interpret that broadly to be

    20    within 24 hours, certainly encompassing 12 hours. 

    21    There is no requirement that the sample tested be

    22    integral to collection, although clearly we prefer

    23    that.  Additionally, if it proves to be infeasible to

    24    test within 24 hours, the regulations provide for the

    25    possibility of exceptions to the regulations, and you

                                                                         70

     1    simply have to request an exemption.  It is under 21

     2    C.F.R. 640.120.  So I think that there really isn't a

     3    regulatory obstacle to doing what is scientifically

     4    and medically appropriate.  We just have to be in the

     5    right dialogue.

     6                CHAIRPERSON HARVATH:  Okay.  On the panel

     7    discussion, the presentations this morning also raised

     8    some very interesting questions that I think would be

     9    good to ask of the panel and get all of your feedback

    10    on for the experiences you have had.  One of the

    11    things I think that will be helpful to us will be your

    12    collective experience on whether this is going to be

    13    a major obstacle and what your experience is with the

    14    testing of the products.  Also, there was early report

    15    in the literature that perhaps some cytokines may

    16    alter some of the test results, and I know that we

    17    have heard this, and I think it would be very

    18    interesting to pose that question to those of you who

    19    have been collecting these products and actually

    20    performing the routine tests on your donors.  So it

    21    would be something very interesting to hear of all the

    22    speakers.

    23                I would like to give everyone an

    24    opportunity to take a 15 minute break and we will come

    25    back here to begin the second part of the morning

                                                                         71

     1    session at 10:00.

     2                (Whereupon, at 9:44 a.m. off the record

     3    until 10:10 a.m.)

     4                CHAIRPERSON HARVATH:  We are going to try

     5    and get started.  Our next speaker, Dr. Dan Ambruso,

     6    is a Professor of Pediatrics and Associate Professor

     7    of Pathology at the University of Colorado Health

     8    Sciences Center, and he is the Associate Medical

     9    Director of Bonfils Blood Center.  It is a pleasure

    10    for me to introduce Dan, and he is going to talk to

    11    you about his experience regarding the functional

    12    properties of granulocytes that he studied from donors

    13    after G-CSF administration.  Dan?

    14                DR. AMBRUSO:  Thank you, Liana.  It is a

    15    pleasure to be here this morning.  I am happy to be

    16    involved in this workshop.  I am going to present to

    17    you some information that we have on normal volunteers

    18    who received G-CSF, and I will say at the outset that

    19    Tom Price has talked about response that his donors

    20    had with a single dose of G-CSF, and I am going to

    21    talk about a project that we were involved with where

    22    our patients received five doses of G-CSF and we

    23    looked before and after the administration and tried

    24    to focus on neutrophil function.  

    25                This seems initially a little bit farther

                                                                         72

     1    away from the practical aspects of blood donors for

     2    granulocytes where a single dose might be more

     3    practical.  On the other hand, I am sure all of you

     4    are aware of the fact, and this was brought up at the

     5    meeting yesterday and it has been brought out in the

     6    literature recently that there may be a number of

     7    paradigms including multiple dose administration of G-

     8    CSF to not only collect stem cells but also

     9    granulocyte support for these patients.  So I think

    10    this information has relevance to granulocyte

    11    collections.

    12                The objective of this talk, as I stated,

    13    is to review detailed studies of neutrophil functional

    14    capacity obtained during G-CSF administration,

    15    multiple dose administration.  I would also, if there

    16    is some time at the end, present some preliminary

    17    findings of functional capacity of neutrophils stored

    18    in the presence or absence of G-CSF.  That was part of

    19    the study as well.  And then I would comment on areas

    20    that we think need further study.

    21                The previous two speakers covered this

    22    part of the talk and I don't need to go into this in

    23    detail.  Everyone knows that the effects of G-CSF

    24    include increased numbers of mature neutrophils

    25    enhanced, and I put that with a question mark because

                                                                         73

     1    I am in agreement and I will show you data to

     2    substantiate this, that neutrophils are really

     3    different.  But the concept of a neutrophil which is

     4    red hot and angry and ready to explode and kill

     5    anything in its path is probably not what we get when

     6    we mobilize and we treat patients with G-CSF.  And one

     7    of the other interesting and important effects of G-

     8    CSF is prolongation of time to apoptosis and its

     9    effect on program cell death.  

    10                Our clinical protocol is summarized here. 

    11    We had healthy adult volunteers and in subsequent

    12    slides I will call these patients.  There were 9 males

    13    and 5 females.  We administered G-CSF at a dose of 10

    14    mcg per kilo subcutaneously for 7 days.  Some of these

    15    patients were part of a control trial for the ACTG

    16    stem cell mobilization study.  We looked and took

    17    peripheral samples or samples of peripheral blood

    18    before the first dose and after the fifth dose of G-

    19    CSF.  In the studies, when you look at the data, day

    20    0 is the first day.  So we actually sampled before the

    21    day 0 dose, and day 4 is when they received the fifth

    22    dose of G-CSF.  In 8 of these subjects, we completed

    23    granulocyte collections after the fifth dose.  The

    24    collections were completed using the COBE Spectra with

    25    hetastarch and the granulocyte products that we

                                                                         74

     1    obtained were stored in the absence or presence of an

     2    added G-CSF 25 nanograms per ml at 22 to 24 degrees

     3    Centigrade in a stationary state.  Samples were

     4    removed from these products at 24 and in some cases 48

     5    hours of storage for analysis.

     6                I will start out with some of the adverse

     7    events.  To summarize, as has been other people's

     8    experience, there were very few adverse events in this

     9    administration.  All of our patients had a mild

    10    headache and bone pain.  We would rate it as grade 1

    11    to 2.  One patient dropped out of the protocol at the

    12    end of the fifth day or fifth dose in apheresis, but

    13    all of the rest had much milder problems.  It usually

    14    peaked by day three of administration and usually

    15    treated well with either ibuprofen or acetaminophen

    16    with resolution of the symptoms.  All of the symptoms

    17    completely resolved within 24 to 48 hours of

    18    discontinuing G-CSF.

    19                Just a few words about the quantitative

    20    response.  As with other studies that have looked at

    21    mobilization of neutrophils with G-CSF, we saw a

    22    marked increase in the leukocyte count from a mean of

    23    4,870 per microliter up to almost 32,000, an almost

    24    tenfold increase in the absolute neutrophil count, and

    25    a marked increase in the percentage of band forms in

                                                                         75

     1    these individuals.

     2                Now I show this slide as a prelude to

     3    reviewing the functional data on the neutrophils. 

     4    Most of you don't need a lecture on neutrophil

     5    function, but I do this to give you a perspective and

     6    a focus on how we organized our function studies. As

     7    you know, neutrophils travel in the laminar flow of

     8    the blood stream until they identify an area of

     9    inflammation, exhibiting a rolling behavior at first

    10    and then finally sit down with firm adherence,

    11    diapedese through the endothelial barrier and move

    12    towards the area of infection or inflammation.  Once

    13    they get there, they ingest the microorganisms, which

    14    does two things.  Associated with ingestion is

    15    activation of the respiratory burst, the neutrophil

    16    NADPH to oxidase enzyme system, which is responsible

    17    for initiation of production of oxygen radicals and is

    18    associated with oxygen dependent killing.  In

    19    addition, there is a variety of contents in the

    20    granules which then are released into the

    21    phagolysosome which affect oxygen independent killing.

    22                So we essentially focused into two general

    23    groups, those kinds of function and biochemical

    24    parameters that are related to adhesion and motility,

    25    and those that are related to microbicidal activity

                                                                         76

     1    and degranulation or the status of the granules.  

     2                I present this also because again of the

     3    concern that over the past 10 years or so, somehow we

     4    have the concept that the neutrophil that is produced

     5    under the influence of G-CSF, this is not only for

     6    donors but certainly for patients who get G-CSF, is a

     7    neutrophil that is charged and ready to go.  And my

     8    concern is, and this certainly has been presented by

     9    Dr. Price in the last talk -- my concern is that in

    10    fact if this were so, we would be in big trouble. 

    11    When the neutrophil is able to get to the site of

    12    infection and to eliminate the organisms, that is one

    13    issue.  If the neutrophil is charged on the

    14    endothelial surface and is activated on the

    15    endothelial surface, you get excessive inflammation

    16    and probably you get -- this is responsible for a lot

    17    of multi-organ failure syndromes which we see

    18    certainly in the lung and perhaps other organs.  So my

    19    concern about this has always been that if the

    20    neutrophils are so charged, we are going to be putting

    21    patients at risk for these multi-organ failure

    22    syndromes.  Certainly that is not borne up in the

    23    patients who have gotten granulocyte transfusions, but 

    24    perhaps the patients who have received granulocyte

    25    transfusions or G-CSF for clinical indications.

                                                                         77

     1                The first thing we did was to look at

     2    chemotaxis.  This was done in a modified Boydian

     3    chamber.  The results are summarized here.  I hope

     4    that is in focus for all of you.  What you can see is

     5    that we looked at non-directed migration towards

     6    buffer and we looked at migration with zymosan

     7    activated serum, which is essentially C5A.  The open

     8    bars are the 0 values and the closed bars are day 4

     9    values for controls and for the patients, that is, the

    10    volunteers who received G-CSF.  And what you can see

    11    here is a marked decrease in the motility in this

    12    Boydian chamber assay in response to zymosan activated

    13    serum.  There is a mild effect, although this isn't

    14    statistically significant, in terms of directed

    15    migration.  So there seems to be in the neutrophils

    16    that are circulating after the fifth dose of G-CSF

    17    administration, there appears to be a decrease in cell

    18    motility.

    19                We looked at two other parameters that are

    20    related to cell motility and might be a reason for the

    21    reduced motility. One is to look at the expression of

    22    CD11B, which is one of the major adhesion proteins for

    23    the neutrophil.  And we looked at the expression of

    24    CD11B in response to 4 ball ester at the concentration

    25    noted here, FMLP, which is a bacterial tripeptide and

                                                                         78

     1    platelet activating factor.  This slide summarizes the

     2    results for day 0 patients and controls and day 4

     3    patients and controls.  And what you can see is that

     4    expressed as a ratio, the stimulated up-regulation of

     5    CD11B was no different in the treated patients.  In

     6    addition, what I don't have here was the baseline

     7    expression of CD11B, which was not increased in the

     8    patients on day 4 of treatment.  So we could not blame

     9    the decrease in motility on a change in perhaps an up-

    10    regulation in CD11B.

    11                One of the other biochemical correlates

    12    related to motility is F-actin assembly and one can

    13    measure this with a dye MBD felacydin.  One of the

    14    other questions we asked was whether there was

    15    something that was different in the modal apparatus of

    16    the cells.  So again we looked at the two groups, the

    17    controls and patients on day 0 and day 4, and this is

    18    a lot of data.  The important thing is to look here. 

    19    This is again an expression of mean channel

    20    fluorescence in unstimulated cells or cells that are

    21    treated with 10-7 molar FMLP.  And what you can see is

    22    a decrease in F-actin assembly that is statistically

    23    significant and we think probably practically

    24    significant too.

    25                So in fact one of the things that we were

                                                                         79

     1    able to find then was a decrease in motility and an

     2    associated decrease in F-actin.  One of the other

     3    things we wanted to look at was channels in cytosolic

     4    calcium.  This ends up being very important in

     5    motility and ingestion and in most receptor mediated

     6    functions of the cell.  In these experiments what we

     7    did was to load the cells with a fluorescent

     8    indicator, binding calcium Endol-1, and we treated the

     9    cells with FMLP.  This is a plot of the results for

    10    one patient.  The neutrophils that were collected on

    11    day 0 were assayed and the calcium flux was followed

    12    in response to FMLP and the same patient on day 4. 

    13    What you can see on day 4 is a marked increase in the

    14    total flux in the cells.  The onset and initial rate

    15    are the same for day 0 and day 4, but this increase is

    16    two to three-fold.  If we look at all of the patients

    17    now, and this slide summarizes results for all

    18    patients, looking at cytosolic calcium, and what I

    19    have here is the response of cytosolic calcium to FMLP

    20    10-7 molar and also platelet activating factor.  There

    21    are two columns for each stimulus.  The first column

    22    reflects the baseline level of calcium and the second

    23    the peak calcium flux.  This is in micromolar

    24    concentration.  What you can see -- the important

    25    thing -- again, a lot of numbers -- the important

                                                                         80

     1    thing is to look at the second and fourth column at

     2    the bottom.  This is the results for day 4.  One can

     3    see a marked increase in all of the patients.  This is

     4    a two- to three-fold increase of cytosolic calcium.

     5                This enhancement in calcium flux in the

     6    cells in response to the specific stimuli, we are

     7    still not sure exactly what that means and the

     8    importance and relevance of this to the chemotaxis and

     9    perhaps other activities is not clear at this point.

    10                So the next group or classification of

    11    studies that we did is spectracidal activity.  This is

    12    a standard bacteriocidal assay.  In these studies,

    13    this summarizes studies for the normal controls and

    14    patients on day 0 which are included in the dots and

    15    in the squares are patients on day 4.  In this assay,

    16    there is a 1 to 1 ratio of bacteria to cells, and the

    17    bacteria is Staph aureus.  This is done in the

    18    presence of 10 percent normal pooled serum.  What you

    19    can see is killing that is equivalent to control in

    20    the patient on day 4.  Perhaps there is a suggestion

    21    of attenuation of killing, but this is not

    22    statistically significant.  

    23                We looked at this in a little bit more

    24    detail because neutrophils when they first get to an

    25    area of inflammation have probably a lot more bacteria

                                                                         81

     1    to phagocytose and kill than just one each.  In

     2    addition, one can see in a variety of patient

     3    disorders a mild killing defect.  So we wanted to

     4    stress the system and we did a killing assay with a

     5    ratio of 10 bacteria to 1 neutrophil.  This is

     6    summarized on the next slide.  What you can see is

     7    that at 30 minutes and at 90 minutes, there is a

     8    statistically different and I think practically

     9    different percent killing in this assay.  So perhaps

    10    there is a mild defect, and I would underscore that --

    11    I would say a mild defect in killing and it may or may

    12    not be significant.

    13                We looked in detail at the respiratory

    14    burst and the oxidase activity.  And I am going to go

    15    through in the next two slides looking at the

    16    respiratory burst measured as cytochrome C reduction

    17    in response to a variety of different agonists.  The

    18    first one we used was FMLP, a chemotractant which at

    19    a little bit higher dose than used in chemotaxis will

    20    activate the oxidase.  On this plot you see the

    21    control and the patients on day 0 and day 4.  What you

    22    can see here is an increase in the FMLP response in

    23    patients on day 4 compared to controls.  This seems to

    24    parallel what was found in vitro and also other

    25    studies that have been done, studies that Dr. Dale

                                                                         82

     1    presented earlier this morning.  

     2                When we look at a variety of other

     3    agonists to try to define or get a complete picture of

     4    the oxidase, we see some divergence in the results. 

     5    These are results for host cell superoxide activity

     6    production with 4 ball ester.  As we can see here,

     7    this is day 0 control and patient and this is day 4

     8    control and patient.  We see a marked depression in

     9    the PMA response.  If you look at another stimulation

    10    sequence, and in this sequence we try to look at

    11    priming of the cells and we essentially prime the

    12    cells or incubate the cells with platelet activating

    13    factor for three minutes and then come back and look

    14    at the response to FMLP.  This usually gives us kind

    15    of the maximum respiratory burst.  This is even a

    16    stronger set of agonists than PMA or most other

    17    agonists that you can use to look at the respiratory

    18    burst.  What you can see is again a decrease or an

    19    attenuation of the production of superoxide in the

    20    intact cells with this stimulus.

    21                Now if you look at a third stimulus, which

    22    is opsonized zymosan, which is essentially a

    23    phagocytic stimulus which is coated with complement

    24    and so ingestion is most likely by complement and C3BI

    25    receptors, what one sees is no difference in the

                                                                         83

     1    respiratory burst.  This is probably a more

     2    physiologic stimulus.  So you see a divergent set of

     3    reactivity that shadow or characterize the respiratory

     4    burst.  Some are increased, some are low, and some are

     5    normal.

     6                We looked very carefully at oxidase

     7    components and this would be -- I am not going to show

     8    you all the data, but this would be of interest to

     9    individuals who are looking at or are interested in

    10    the oxidase itself.  What we found on these cells when

    11    we looked at subcellular fractions, we found increased

    12    amounts of cytochrome B558 in the plasma membrane and

    13    normal contents of the cytosolic oxidase components,

    14    the P47-phox, the P67-phox and the P40-phox.   So the

    15    oxidase itself seems to be intact, but we seem to have

    16    to certain kinds of stimuli a decreased response,

    17    which would suggest that it is other systems perhaps

    18    than the structural oxidase proteins that are

    19    affected.

    20                One of the other things that we did was to

    21    look at granular marker proteins in order just to

    22    define the status of granules in these cells.  As Dr.

    23    Dale suggested earlier, these cells look different. 

    24    If you do EMs, you have a sense looking at the EMs

    25    that the granules are not quite the same and the

                                                                         84

     1    granule compartments are not the same.  So we looked

     2    at this and looked at alkaline phosphatase, which is

     3    increase, which everybody would expect and which has

     4    been really well documented as an effect of G-CSF on

     5    neutrophils.  Myeloperoxidase seems to be normal.  The

     6    specific granule marker, lactoferrin, seems to be

     7    decreased.  The question that is raised by this data

     8    as to whether there is a defect in the specific

     9    granules themselves and their production, when we

    10    looked at cytochrome B, the content of cytochrome B

    11    was actually normal to increased.  So this needs to be

    12    looked at a little bit more carefully.  I am not sure

    13    at this point that we can say that there is a decrease

    14    in specific granules, but there appears to be a

    15    decrease in specific granule content in some proteins.

    16    And that may certainly have some functional impact on

    17    the cell.

    18                One interesting side note, and that is 

    19    that we have saved now cell lysates and subcellular

    20    fragments of plasma in the membrane and granules, is

    21    to look at also to save RNA, and the question is what

    22    does RNA do to some of the genes and some of the

    23    proteins in the cell.  The interesting observation

    24    that we made is that when one looks at the cells that

    25    are collected on day 4, there is a much larger amount

                                                                         85

     1    of RNA which can be extracted.  This is roughly the

     2    RNA content for 108 cells.  This is control day 0 and

     3    patient day 0 and control day 4 and patient day 4. 

     4    You see almost a two-fold increase in RNA.  In fact,

     5    when you store cells for 24 hours, you double the RNA

     6    again in looking at how much you can extract from the

     7    cells.  This is very interesting.  We are not sure

     8    what the significance of that is, but I think it is  

     9    going to be an important clue to some of the defects

    10    that we are finding.

    11                This is just kind of a mental break.  I

    12    wanted to talk a little bit about apoptosis, because

    13    this is another area that we evaluated in this study

    14    with patients.  These tests are done by looking

    15    morphologically.  We take cells at the sampling times

    16    and we isolate them and we put them into culture with

    17    RPMI and fetal calf serum.  Then at different times

    18    after that, up to 48 hours, we take little samples out

    19    and we evaluate them for the extent of apoptosis.  Of

    20    course these are very labor intensive studies and

    21    people go crazy as they stay up in the lab and try to

    22    get these things done.  So our argument was, was this

    23    a-pop-tosis or apop-tosis.  I mean, you get pretty

    24    crazy when you are doing these kinds of experiments. 

    25    Dr. Levy, who is from Dublin, Ireland, was a fellow in

                                                                         86

     1    my lab who was involved with most of the studies that

     2    you are seeing here.   He had a different feeling and

     3    he would come out with his thick Irish accent and say,

     4    no, it is not any of those, it is really O'potosis.

     5                Let me show you an example.  This was a

     6    technique in which you use a double stain, preputium

     7    iodine and acridine orange.  These are both non-

     8    apoptotic cells.  You don't see any of the nuclear

     9    changes related to apoptosis.  This is a dead cell and

    10    this is a live cell.  This is a live apoptotic

    11    neutrophil.  So on the basis of these morphologic

    12    features, we would characterize during the culture the

    13    percentage of cells that were apoptotic, and we could

    14    generate a graph, if you will.  We called it the LT50. 

    15    It probably should be the AT, the apoptosis time 50,

    16    but the time to 50 percent apoptosis.  That is what is

    17    graphed here.  What you can see is the dark bars are

    18    the patients at day 0 and day 4 with the controls. 

    19    What you can see is that before G-CSF administration

    20    to the patients or in the control group, we see a time

    21    to 50 percent apoptosis, live apoptosis, is somewhere

    22    around 17 hours.  If you look at patients on day 4

    23    after the fifth dose of G-CSF, what you see is the

    24    time to 50 percent apoptosis is prolonged to about 34

    25    hours, it is doubled.

                                                                         87

     1                In other studies to the in vitro system,

     2    we added G-CSF to a dose of 25 nanograms per ml.  What

     3    we saw is that these cells could respond further by

     4    prolonging their apoptosis.  You will notice that this

     5    curve is now a different curve starting out at 30

     6    hours and what we see is a prolongation of the control

     7    and patient day 0 and control day 4 cells to somewhere

     8    around 42 to 45 hours.  And you see the patient day 4,

     9    which had received 5 doses of G-CSF, is prolonged even

    10    further.  So the cells have, at least in vivo

    11    circulating that we can take out and culture and look

    12    at apoptosis, have something going on that prolongs

    13    the process to apoptosis and that these cells can be

    14    further manipulated by adding G-CSF in vitro to

    15    prolong that time to apototic death even further.  So

    16    this is actually the good news about G-CSF.  This is

    17    not doctor-assisted suicide of the cells, but in fact

    18    the reverse, that is, we can help prolong the lifespan

    19    of the cells.  That has, I think, some implications

    20    perhaps for storage.

    21                So in summary, administration of G-CSF for

    22    five consecutive days is well tolerated with, I think,

    23    minor problems and adverse events.  Although I would

    24    echo Tom's comments about how are you going to get

    25    normal donors to accept any kind of discomfort which

                                                                         88

     1    may be significant.  And certainly we can see an

     2    enhanced number of mature neutrophils.  I haven't

     3    shown you this, but I will present a summary slide in

     4    a minute where you certainly get robust huge numbers

     5    of granulocytes that you can collect by apheresis. 

     6    Interestingly, the time of apoptosis is delayed and

     7    that the neutrophils themselves that are mobilized

     8    under the influence of G-CSF continuous administration

     9    for five days present a divergent pattern of

    10    functional characteristics but overall the function is

    11    not markedly enhanced.  And we would further summarize

    12    that we think the effect of prolonged administration

    13    of G-CSF and the advantage to patients themselves may

    14    be related more to numbers and the effect on apoptosis

    15    and perhaps survival and not so much the enhanced

    16    functional characteristics.

    17                Let me just summarize some of the results

    18    we have in storage.  We can perhaps talk about this

    19    this afternoon in the discussion section and the

    20    poster session.  But as I said, there were 8 products

    21    that we stored with this in the presence and absence

    22    of G-CSF.  What we noticed is that looking at

    23    superoxide and intact neutrophils to all the stimuli

    24    that I mentioned before didn't seem to change much

    25    during the first 24 hours of storage and then it

                                                                         89

     1    deteriorated gradually by 48 hours.  It was about half

     2    of what you saw that I had presented with the time 0

     3    studies.  Most of the collected neutrophils remained

     4    viable, that is, greater than 98 percent viability of

     5    the neutrophils in storage, and were not apototic at

     6    that time.  Their apototic rate was probably no

     7    different than what we found for the neutrophils that

     8    were collected right at the time or just before we had

     9    done the granulocyte collections, and that is the  34

    10    hour time.  So there seems to be, at least for 24

    11    hours and it may be longer, this is something we need

    12    to look at a little bit more carefully, a viability

    13    and postponement of apoptosis.  Chemotaxis, although

    14    it was deficient -- as I showed you, the day 4 data

    15    was deficient -- in storage this didn't get any worse,

    16    at least for the first 24 hours of storage.  And when

    17    we looked at all these things and the addition of 25

    18    nanograms per ml of G-CSF, we really didn't change

    19    these characteristics of the neutrophils.  That is

    20    probably most likely that we didn't add enough and

    21    that may need to be looked at a little bit more

    22    carefully.  But there may not be really any additional

    23    effect that these cells have.

    24                So what are considerations for future

    25    studies?  First of all, I think more work needs to be

                                                                         90

     1    done in defining standardized schemes for mobilization

     2    and collection.  There may be reasons -- certainly one

     3    can collect granulocytes after a dose of G-CSF, but

     4    there may be reasons to look at multiple doses of G-

     5    CSF.  There may be reasons to perhaps think about

     6    other cytokines as well.  So this needs to be defined

     7    a little bit more clearly.  It would be worthwhile to

     8    develop the optimal storage conditions for neutrophils

     9    to try to support normal function and survival for a

    10    little bit longer.  Right now what we are doing, as

    11    Dr. Price has suggested -- and we have a difficult

    12    time in the blood bank because we get our donors in

    13    several days early and use that sample to allow the

    14    physicians to get the blood, that is, we can release

    15    it with that.  We also have to do the processing on

    16    that sample that we collect.  So this is really

    17    problematic.  Obviously in terms of providing a

    18    product that has no storage time, if we could develop

    19    some, even for 48 hours, it would be very helpful in

    20    doing the collections.  We really need to expand and

    21    develop techniques for evaluating in vivo function of

    22    transfused granulocytes.  To extend in vitro studies

    23    to evaluate this and to look at what their functions

    24    are in the patients is going to be very, very

    25    important.  And, of course, clinical trials to

                                                                         91

     1    document their efficacy, toxicity, and cost

     2    effectiveness are going to be important to complete in

     3    order to really revitalize this type of blood

     4    component.  So with that I will stop.

     5                CHAIRPERSON HARVATH:  Thanks, Dan.  The

     6    next speaker is Dr. Susan Leitman.  Dr. Leitman is the

     7    Chief of the Clinical Services Section of the

     8    Department of Transfusion Medicine here at the NIH. 

     9    She is going to talk to us about her experience with

    10    G-CSF mobilized granulocytes.

    11                DR. LEITMAN:  Thank you, Liana.  And thank

    12    you for inviting me to speak at today's conference. 

    13    This is a slide I made to entitle a talk at another

    14    conference on this topic, and I found that it applied

    15    well to the issues that we are bringing before the FDA

    16    perhaps in consideration of licensure of this product. 

    17    And with growth factor mobilized granulocytes, is this

    18    an exciting or stimulating -- no pun intended -- new

    19    component or are we stuck with the same old problems.

    20                I would like to remind all of us, as if we

    21    needed to be reminded, that despite nearly three

    22    decades of clinical experience and dozens of

    23    publications of observational or controlled studies,

    24    the FDA does not recognize granulocyte concentrates as

    25    an approved blood component. And from the very nice

                                                                         92

     1    review we heard by Dr. Dale this morning, there are

     2    very good reasons for that non-recognition.  This is

     3    my version of Ron Strauss's review from the Blood 1993

     4    article reviewed by Dr. Dale this morning.  I want to

     5    point out that the reason we can't find efficacy

     6    across all of the seven prospective, some randomized

     7    and some non-randomized studies, is in large part due

     8    to the choice of the subjects for the study, and thus

     9    in studies designed to determine clinical efficacy you

    10    have to choose patients in whom the mortality is

    11    estimated to be substantially above 60 percent.  In

    12    those studies in which the survival was 60 percent or

    13    greater, no efficacy could be demonstrated.  It was

    14    only when the mortality was quite high that efficacy

    15    could be demonstrated here, with mortality of percent

    16    surviving of 26, 15, and 36 percent.  So in designing

    17    prospective trials, we have to choose the right

    18    population to study and, as Dr. Dale already stated,

    19    have sufficient numbers to power the study or

    20    analysis.

    21                I will talk just for a moment about dose

    22    because that has been covered very well so far this

    23    morning.  To remind you that granulocyte apheresis

    24    yields without any donor preparation are in the range

    25    of .3 to .5 times 1010 cells.  With the addition of

                                                                         93

     1    hydroxyethyl starch, this is high molecular weight

     2    starch, that increases or that doubles to .5 to .9

     3    times 1010.  When one uses some combination and some

     4    regimen of steroid administration plus starch, that

     5    again doubles to 1 to 2 times 1010.  I am going to

     6    diverge for a moment and say that granulocyte

     7    apheresis took a giant step backwards in the mid to

     8    late 1980's with the introduction of a new form of

     9    starch, pentastarch.  Pentastarch is a less highly

    10    substituted amylopectin backbone, the same backbone

    11    that is in hetastarch, with an average molecular

    12    weight of 264 rather than 480,000, a significant

    13    reduction in the number of hydroxyethyl groups per

    14    glucose residue.  What made pentastarch very

    15    attractive to blood bankers was the safety for the

    16    donor in that the 24-hour urinary excretion is much

    17    higher with pentastarch than hetastarch and the

    18    overall survival in blood is only 96 hours as opposed

    19    to 17 to 26 weeks with some residual hetastarch

    20    remaining in a donor's body for substantial periods of

    21    time.  So the entire blood banking field sort of moved

    22    to pentastarch rather than hetastarch in the late

    23    1980's.  But if you look at the studies of

    24    pentastarch, the efficacy of granulocyte apheresis or

    25    the efficiency of granulocyte apheresis and the yields

                                                                         94

     1    were never prospectively compared to hetastarch.  A

     2    fellow in our lab, Dr. John Lee, who is now with the

     3    FDA, did a very nice set of studies in the early

     4    1990's where he looked at the comparison of

     5    pentastarch and hetastarch.  Just to remind you, the

     6    granulocyte collection efficiency with apheresis

     7    devices, the GCE, varies directly in proportion to the

     8    donor's erythrocyte sedimentation rate.  The more

     9    quickly the red cells sediment, the better the

    10    separation in the granulocyte layer and the more

    11    efficiently the machine can collect them.  So with

    12    increasing donor sedimentation rates, there is an

    13    increasing granulocyte collection efficiency.  What

    14    was not known at that time or not clearly defined was

    15    that hetastarch quadruples the donor's sedimentation

    16    rate in vivo and pentastarch increases it by one and

    17    a half to two-fold.  

    18                When John Lee prospectively compared in 72

    19    apheresis donors a granulocyte apheresis procedure

    20    using pentastarch and three months later a granulocyte

    21    apheresis procedure using hetastarch and looked at the

    22    granulocyte collection efficiencies, if they were the

    23    same the line of identity would be here shown by this

    24    dash blue line, and they were not the same.  The

    25    granulocyte collection efficiency with hetastarch was

                                                                         95

     1    substantially and significantly better than with

     2    pentastarch in all but three donors.  If you look at

     3    the yield, not only the GCE, you see again here is the

     4    line of identity and with hetastarch the yields were

     5    always, except for three donors, substantially greater

     6    than with pentastarch.  I will summarize this

     7    numerically on the next slide.  So there is a 60 to 70

     8    percent increase in granulocyte yields times 1010. 

     9    This is before G-CSF.  These are all non-G-CSF

    10    mobilized donors.  From 1.4 to 2.3 times 1010 in these

    11    72 paired collections.  Collection efficiency

    12    increases from 33 to 58 percent.  With the publication

    13    of this study, I believe that most centers have

    14    returned to hetastarch.

    15                At about the same time that Dr. Dale was

    16    giving his five college students various doses of G-

    17    CSF, we were giving 20 healthy apheresis donors under

    18    protocol three varying mobilization regimens.  The

    19    donors underwent three leukopheresis procedures each

    20    separated by at least four weeks.  The three

    21    preparative regimens were nearly identical to what you

    22    have heard this morning -- dexamethasone 8 mg orally

    23    was given 12 hours prior to donation.  We would tell

    24    our donors to take the dexamethasone about one hour

    25    after dinner, which is about 8 p.m., and that is 12

                                                                         96

     1    hours before they come to our apheresis center at 7:30

     2    to 8:30 in the morning.  We give them the next dose of

     3    dexamethasone to take home with them at the time of

     4    apheresis so that they don't have to come to a

     5    pharmacy to get the tablets.  G-CSF we administered at

     6    a dose, a per kilogram dose of 5 mcg per kilogram, and

     7    we used Amgen's form of filgrastim, and we gave it

     8    subcutaneously between 16 to 24 hours prior to

     9    donation.  And then in the third arm, they received

    10    both types of preparative drugs.  

    11                G-CSF comes commercially or is available

    12    commercially in two size vials, a 300 mcg vial and a

    13    600 mcg vial, 300 per ml.  So it is one ml in the

    14    first vial and 2 ml in the second vial, which is

    15    exactly why in the Seattle study they used either 300

    16    or 600.  Apparently the Government on GSA schedule

    17    gets a vial that contains 480 mcg, which is partly why

    18    we use 600 mcg per kilo, because that does not exceed

    19    one 480 mcg vial.  So this is going to be a little bit

    20    different in comparing our study to the Seattle

    21    studies, but probably not substantially so.  

    22                All our granulocyte procedures are

    23    performed using a Baxter CS-3000 plus apheresis

    24    device, continuous flow, two access sites needed. 

    25    These are the parameters of leukopheresis.  The two

                                                                         97

     1    instrument chambers who blood flow rate is between 50

     2    and 60 ml per minute.  Our endpoint volume

     3    traditionally in the last 15 years of collecting

     4    granulocytes has been 7 liters.  So for this study, we

     5    did not change that.  You will note the Seattle

     6    protocol was 10 liters and other protocols vary from

     7    7 to 12 liters processed.  The anticoagulant is sodium

     8    citrate.  The sedimenting agent is 6 percent

     9    hetastarch. 

    10                If you read the operating manual for

    11    performing this procedure on the CS-3000, it tells one

    12    to set the interface offset at 15.  No one does that. 

    13    In studies done 10 years ago along with the engineer

    14    who developed this device, Mr. Herb Cullis, it was

    15    found that an interface offset setting of 33 yields

    16    optimal efficiency of the procedure, and so most of us

    17    have been using an IO of 33 for the past decade, not

    18    what is in the operating manual.  

    19                These are the results of the three

    20    different regimens, dexa, G-CSF, and D+G.  This is the

    21    peripheral blood polymorphonuclear count immediately

    22    prior to apheresis, and you can see that the addition

    23    of G-CSF or D+G increases the white cell

    24    polymorphonuclear cell count in the donor by 3.5 to

    25    4.5 fold, very similar to what you saw this morning,

                                                                         98

     1    from 6,000 to 21,000 to 29,000.  Similarly, the

     2    product content increases 2.5 to 3.5 fold from 2.5

     3    times 1010 with our traditional dexamethasone alone

     4    arm to 5 to 7.2 times 1010 with a combination of both. 

     5    All of these comparisons are statistically significant

     6    at the .05 level for every comparison within groups.

     7                Addition of dexamethasone to G-CSF alone

     8    resulted in a 43 percent increase in the granulocyte

     9    yield in the product.  We also looked at granulocyte

    10    collection efficiency and our usual efficiencies, as

    11    you have seen in the previous slide, are in the range

    12    of the low 60 percent.  Somewhat to our surprise, we

    13    found that the efficiency dropped by 10 percent when

    14    we added G to this regimen.  When we spoke with the

    15    engineer who had designed this device, he told us that

    16    the machine was designed for donor counts of 10,000,

    17    not for total white counts of 30,000 to 35,000, and

    18    that was necessary probably was a complicated change

    19    to the interval between spillovers and the duration of

    20    spillovers, for those of you used to this machine, and

    21    that we would eventually do that.  But as a start, we

    22    simply increased the interface offset setting to 45,

    23    and I don't have those numbers right here because they

    24    haven't changed.  Simply increasing the depth of

    25    penetration into the buffy coat layer as this machine

                                                                         99

     1    collects the cells did not significantly increase the

     2    GCE.  So I think further work needs to be done with

     3    this particular device in maximizing collection

     4    efficiencies.  

     5                One day, we just happened to have three of

     6    these products quite by accident in the processing

     7    area of the transfusion medicine department, and they

     8    looked so distinctly different that we took the

     9    opportunity to take a picture of them.  This is the

    10    traditional dexamethasone stimulated product.  It

    11    looks redder because the buffy coat layer is less

    12    thick.  This is G-CSF alone with a thicker buffy coat,

    13    and this is the combination of G-CSF plus

    14    dexamethasone.  This is just sedimentation on the

    15    counter top over the course of the six to eight hours

    16    between the end of collection and the time of

    17    transfusion.  You can see the buffy coat layer

    18    sediment out.  In our institution, we start apheresis

    19    in all such donors on this protocol at 8:00 to 8:30 in

    20    the morning.  The product comes off the machine by

    21    10:30 in the morning, and we do same day transfusion

    22    transmitted infectious disease testing starting at

    23    9:00 in the TTV laboratory.  So we do same day testing

    24    although we are one of the very, very few institutions

    25    that I think can continue to do that today.  So our

                                                                        100

     1    products are transfused at 8:00 p.m. that day.  They

     2    spend about 10 hours on the shelf and we saw the

     3    sedimentation within those 10 hours.

     4                What is the effect on the product of the

     5    three different regimens other than in the

     6    polymorphonuclear leukocyte count?  We always process

     7    the same volume.  This is the volume without the

     8    anticoagulant added.  So this is actually actual true

     9    blood volume processed.  The machine is set to 7

    10    liters but about 500 ml of that is anticoagulant.  The

    11    product volume is set by the operator. We

    12    traditionally set it to be about 240 ml.  The platelet

    13    content was identical across all three collection

    14    regimens and the red cell content was identical across

    15    all three collection regimens.  Just to remind you,

    16    the mean red cell content is about 30 ml of packed

    17    cells, so a cross match is always necessary between

    18    donor and recipient.

    19                This is a summary that I made a while ago

    20    and it is not updated to reflect the newer Seattle

    21    data, which we heard this morning and which came out

    22    in August.  But it looks at the four published studies

    23    at that time of granulocyte apheresis yields following

    24    G-CSF containing regimens.  The dose varied from 200

    25    mcg per day to 5 mcg per kilo.  Steroid use was

                                                                        101

     1    variable.  Starch use was variable, pentastarch or

     2    hetastarch.  Volume processed was variable.  And the

     3    yields at that time were in the range of 4 times 1010

     4    or 40 times 109, except for our experience in which it

     5    was double and the current Seattle experience in which

     6    it is right about this number -- 82 is what we heard

     7    this morning.  I think the difference here was in the

     8    steroid use.  In these initial studies, we were always

     9    using steroids in our clinical study in products that

    10    were getting administered to patients and we were

    11    always using hetastarch, whereas the type of starch

    12    used and steroid use varied.

    13                I show this slide because it wasn't quite

    14    presented in this way by the earlier speakers today. 

    15    This is the adverse reactions to mobilization regimens

    16    in donors who have undergone all there preparative

    17    regimens.  With dexamethasone alone, we have an n of

    18    38.  And 44 percent of donors have symptoms and they

    19    are almost all universally related to hyperactivity --

    20    insomnia, feeling wired, feeling restless, waking up

    21    at night several times.  And a small proportion had

    22    flushing and this was commonly delayed.  So they would

    23    call us from work later that day to say their

    24    colleagues told them their face and their ears were

    25    bright red.  It could also even happen the next day

                                                                        102

     1    after.  So 56 percent of such donors on dexa alone did

     2    not have symptoms.  With G-CSF alone, the same number,

     3    n equals 38.  We had the usual 39 percent with bone

     4    pain, 26, a quarter, with headache, 24 percent with

     5    that sense of wiredness, and also 5 percent with

     6    flushing.  Only 32 percent did not have any symptoms

     7    at all.  With the combination, the instance of bone

     8    pain is a little bit higher.  Headache is about the

     9    same.  Insomnia is the same with dexamethasone alone. 

    10    Some nausea.  I forgot to mention the fatigue.  10

    11    percent have fatigue whenever you give G-CSF and

    12    flushing, so that only 28 percent did not have

    13    symptoms.

    14                We have enrolled 120 donors on this study,

    15    similar to what Dr. Price described this morning.  We

    16    took them from our pedigreed platelet pheresis donor

    17    population.  And as he told you this morning, when you

    18    approach these intensely altruistic individuals with

    19    yet another regimen or a new product that may increase

    20    the potential for survival for critically ill patients

    21    with cancer and other serious illnesses, they are most

    22    eager to cooperate and be, if you will, on the cutting

    23    edge of transfusion medicine practice.  Of all the

    24    reactions I described on the last slide, 15 percent

    25    were judged by the nurses or myself in asking the

                                                                        103

     1    donors these questions to be severe and interacting

     2    with everyday activities of the donor.  8 of 76 of our

     3    first donors or 10 percent -- and this continues, it

     4    is now 10 out of 100 -- have requested discontinuation

     5    of G-CSF mobilized collections.  The most common

     6    reason was they didn't like feeling as if they had the

     7    flu once a month -- or as one donor puts it, I am

     8    tired of aging from age 40 to age 80 overnight once a

     9    month.  The other common reason was the inconvenience

    10    of coming to the blood bank twice, once the day before

    11    the injection and once to donate.  Donor reactions

    12    were stereotypic.  Mild reactions tended to become

    13    milder with further donations.  An initial 10 percent

    14    of donors had no symptoms on subsequent G+D

    15    mobilization although they had had symptoms on their

    16    first occasion.  

    17                Now I would like to talk for the remaining

    18    time on patient outcomes.  The results in the first

    19    three patients that we transfused with G-CSF mobilized

    20    products were so dramatic that as my colleague Harvey

    21    Alta says, you should make slides of things that work

    22    before you find that they don't work.  Make your

    23    slides quickly.  So we made these of the first three

    24    patients.  Our very first patient was a 55 kilo female

    25    with T cell large granulolymphocytic leukemia who had

                                                                        104

     1    a sigmoid phlegmon due to diverticulitis.  She had a

     2    bacterial process.  She was extremely ill and toxic,

     3    persistent rigors, chills, fevers, unresponsive to

     4    antibiotics.  The second patient was a larger male,

     5    130 kilo, day 10 post a T cell depleted marrow

     6    allograft for myeloma with a systemic Aspergillus

     7    flavum infection.  The third patient was a similar day

     8    7 post T cell depleted marrow allograft for CML with

     9    a systemic fusarium infection. 

    10                Let me go back before I do that.  What was

    11    dramatic in all these patients was the almost

    12    immediate response to the administration of these

    13    cells.  This patient became afebrile for the first

    14    time the day of the granulocyte administration and

    15    remained afebrile until she actually recovered her own

    16    white count.  Both of these patients were showering

    17    skin with new systemic fungal lesions on a daily or

    18    more often than daily basis.  One could watch the new

    19    lesions develop.  New lesions stopped developing with

    20    the first granulocyte transfusion and old lesions

    21    resolved with subsequent granulocyte transfusions. 

    22    This is the saw-tooth pattern you see when you look at

    23    the increment in ANC, absolute neutrophil count, with

    24    each subsequent granulocyte transfusion.  These are

    25    the first two patients, who as I said had very

                                                                        105

     1    dramatic responses.  The first got a total of four

     2    granulocyte transfusions.  Her absolute neutrophil

     3    count was 0, increased to 2, and as has been said by

     4    speakers this morning, remained elevated for the next

     5    8 hours, which we had never seen before.  So the next

     6    day she gets another increment of 2,000 and goes up to

     7    4,000.  By the next day, she gets another increment

     8    and goes up to 6,000.  We do not collect on the

     9    weekends unless it is a very serious patient problem. 

    10    So we did not collect on this day and she promptly

    11    dropped her neutrophil count at 30 hours.  We gave her

    12    one more transfusion and then you can see her own

    13    cells recover.  

    14                This is the recipients of the T cell

    15    depleted marrow allograft.  They increment to 1,000

    16    and stays there for 8 hours and increments to 3,500. 

    17    Then the weekend occurs and we wanted to see if he had

    18    recovered his own counts.  He had not.  The same saw-

    19    tooth pattern, the weekend, and then his own counts

    20    finally returned.

    21                We looked at the one-hour post transfusion

    22    increment in the ANC level as a function of the number

    23    of granulocytes transfused for these first three

    24    patients and there was a direct correlation, which was

    25    highly significant, with the increasing counts as a

                                                                        106

     1    function of the number of granulocytes transfused. 

     2    This graph is perhaps less steep because there were

     3    only four points.

     4                How did they do overall, though?  They

     5    clearly responded in terms of their infection in the

     6    short-term.  The first patient is in complete

     7    remission and back to her everyday activities now. 

     8    Her diverticular phlegmon was removed surgically after

     9    her own white count recovered.  The second patient

    10    died two months after the course of granulocyte

    11    transfusions due to multi-organ system failure as a

    12    complication of bone marrow transplant.  Aspergillus

    13    was present at autopsy but was not thought to be

    14    contributory to his death.  The third patient

    15    stabilized, eventually was discharged from the

    16    hospital and is still alive now with chronic graft

    17    versus host disease.  Serial one-month skin biopsies

    18    were obtained and at day 30 and 60, he still had

    19    fusarium in his skin, but at day 100, the fusarium was

    20    gone.  So it takes a long time to clear invasive

    21    filamentous fungus infection.

    22                Here is our summary of the 14 total

    23    neutropenic recipients of G-CSF mobilized granulocyte

    24    transfusions to date at the NIH Clinical Center.  You

    25    heard about the 19 patients at the Fred Hutch, and the

                                                                        107

     1    data are very, very similar.  These 14 individuals

     2    received a total of 135 granulocytes that had been

     3    mobilized with G-CSF plus dexamethasone.  In some

     4    cases I have the total number of G-CSF mobilized

     5    granulocyte transfusions in parenthesis because the

     6    total reflects both dexa and G-CSF mobilized products. 

     7    We have given this product to 4 patients with aplastic

     8    anemia.  Three of them had invasive fungus infections,

     9    Aspergillus or fusarium, and one had a strep

    10    pneumonia.  All four patients initially improved. 

    11    These two stabilized to an impressive degree.  The

    12    fusarium resolved completely.  That was the previous

    13    patient.  And the strep infection or the pneumonia

    14    promptly improved.  However, in the first two

    15    patients, eventually since they didn't recover their

    16    own counts, they had ANCs of close to zero and the

    17    disease progressed.  In our experience here, we give

    18    immunomodulatory therapy with ATG and cyclosporin or

    19    cyclophosphamide.  It takes 6 weeks to see an increase

    20    in the ANC with about 65 to 75 percent of all patients

    21    responding, and 6 weeks of an ANC of zero, even in the

    22    presence of granulocyte transfusions, is very

    23    problematic.  So only one was discharged from the

    24    hospital.  

    25                In the allo-peripheral blood stem cell

                                                                        108

     1    transplant patients, there were four, three had

     2    disseminated fungal  infections and one had an RSV

     3    pneumonia, which we didn't know at the time.  We

     4    thought he had a fungal infection or a fungal

     5    pneumonia.  These two patients -- this patient was

     6    started when he was nearly in extremis.  The

     7    Aspergillus was progressive and he rapidly died.  This

     8    patient continued to do poorly despite granulocyte

     9    transfusions until we realized he had RSV as his main

    10    process and not fungus.  These two patients improved. 

    11    I discussed them on an earlier slide.  One died of

    12    multi-organ system failure unrelated to fusarium.  One

    13    is doing well.  This patient with a non-Hodgkin's

    14    lymphoma and two patients with NHL had a vancomycin

    15    resistent enterococcus and multi-organ system failure

    16    and was an extremis when we started granulocyte

    17    transfusions, had progressive disease and died. 

    18    Another young girl with pulmonary Aspergillus in the

    19    setting of HIV infection and non-Hodgkin's lymphoma

    20    had a very nice response to 10 granulocyte

    21    transfusions.  And we have two patients with LGL

    22    lymphoproliferative disorder, both of whose

    23    infections, one bacterial and one candida, resolved. 

    24    And a breast cancer patient with a pseudomonas ulcer

    25    of a myocutaneous reconstruction flap within two days

                                                                        109

     1    worth of transfusions, her own count had returned and

     2    she improved.  I am not sure we can say much about

     3    this one patient in blast crisis at CML who had a

     4    presumed fungal pneumonia who also improved, although

     5    he eventually died of other complications.

     6                So there were 9 of 14 patients with

     7    invasive fungal infections.  Overall, 11 of 14

     8    improved and it was not surprising that these two

     9    patients in extremis and the one patient with RSV

    10    pneumonia did not improve.  But the overall hospital

    11    discharge was slightly less than half as you just saw

    12    from the Seattle experience, and that has been the MD

    13    Anderson experience as well.

    14                These are essentially 14 anecdotes.  And

    15    what we have heard this morning for what we really

    16    need is a randomized prospective trial so that they

    17    become more than just anecdotes.

    18                We have also in this institution treated

    19    a number of patients with chronic granulomatous

    20    disease of childhood, an inherited disorder where

    21    granulocytes cannot make phagocyte oxidase and

    22    membrane bound oxidase, and they can't handle various

    23    types of fungal and bacterial infections and are

    24    subject to recurrent life-threatening infections with

    25    organisms like Serratia, Pseudomonas, Candida,

                                                                        110

     1    Aspergillus, and nocardia.  We have treated a total of

     2    10 such patients with 220 transfusions in the past two

     3    years.  Again, this is a set of 10 anecdotes.  And in

     4    the absence of a prospective trial, there is not that

     5    much that can be said except that 9 out of the 10 had

     6    resolution of infection and 9 out of the 10 were

     7    discharged from the hospital.  And whenever you see

     8    such an excellent response, it makes you think that

     9    perhaps the granulocytes were involved in this

    10    excellent response.  And what makes me have some

    11    confidence in saying that was that in one patient here

    12    and one patient here, they only received two

    13    granulocyte transfusions before an anamnestic response

    14    in their HLA alloantibodies became clear and they had

    15    very significant pulmonary transfusion reactions.  We

    16    stopped the transfusions after two and three

    17    transfusions, but they still resolved their fungal

    18    pneumonias and their bacterial pneumonias with

    19    excellent antimicrobial therapy. So again, in the

    20    absence of a trial, it is hard to say exactly what the

    21    role of granulocytes, even G-CSF mobilized

    22    granulocytes, is in patients with CGD, although there

    23    is some controversy among the clinical care staff, Dr.

    24    Malik and colleagues here at the Clinical Center,

    25    about the role of granulocytes.  

                                                                        111

     1                This is all 14 of our neutropenic

     2    patients.  These are the white cell increments.  This

     3    is the incrementing count, not the absolute count in

     4    that patient -- but the increment following

     5    transfusions of G-CSF mobilized granulocytes.  On the

     6    x axis are hours after transfusion, and please notice

     7    this is not a linear scale.  There is pre-transfusion,

     8    1 hour, 4 hours, 8 hours, 24 hours, and 30 hours.  And

     9    there aren't points at each of these time intervals. 

    10    There is always a point at 1 hour, 8 hours, and 24,

    11    but not always at 4.  The orange line is 10 patients

    12    who did not have either HLA alloimmunization or

    13    splenomegaly.  The 1 hour post increment was 1,900,

    14    very close to the 2,600 that you heard this morning

    15    from the Seattle group.  And 4 hours later in most

    16    patients, that count was slightly higher.  It was

    17    2,000.  Suggesting that one-hour post-transfusion,

    18    there may be some sequestration in organs such as the

    19    spleen and the peak increment is not seen until 4

    20    hours.  There is still an increment at 8 hours, a

    21    substantial increment over the pre-transfusion count. 

    22    That persists at 24 hours and still persists at 30

    23    hours to a very low level.  If you try and calculate

    24    an in vivo or biologic half life, half of 2,000, you

    25    get about 20 hours, which is exactly what Dr. Dale

                                                                        112

     1    reported from his study of radio-labeled cells,

     2    autologous cells, in study participants.

     3                I separated out the three patients with

     4    splenomegaly, two with non-Hodgkin's lymphoma and one

     5    with CML because they had a markedly different

     6    response.  The 1 hour post increment was in the 300

     7    range, went down to the high 200 range, and then

     8    slowly decreased and then went down to 0 at 30 hours. 

     9    And then there was one patient who we did not know had

    10    HLA allosensitization until the first transfusion was

    11    given.  He also did not respond as did the mean group

    12    without HLA allosensitization.  But interestingly, he

    13    had some increment.  It wasn't 0.  That is an n of 1,

    14    so I can't say too much about the amount of increment

    15    you can expect in an allosensitized recipient.  He had

    16    a multi-specific positive lymphocytotoxicity screen. 

    17    So this was a very impressive alloimmunization in

    18    vitro.  This individual was intubated and had

    19    monitoring of every possible pulmonary parameter in

    20    the ICU, and we looked carefully at whether there was

    21    02 desaturation, increased need for positive index

    22    pressure, decreased compliance of the lung, increased

    23    temperature, chills, et cetera, related to the

    24    transfusion of HLA incompatible very large numbers of

    25    granulocytes and we did not see it in this n of 1.  

                                                                        113

     1                There was one patient who received a total

     2    of 11 granulocyte transfusions early in the course of

     3    our work with G-CSF mobilized products, and on only

     4    five of those occasions were we able to find a donor

     5    enrolled in the protocol that could give G-CSF

     6    mobilized product.  On the other six occasions, she

     7    received a dexa mobilized product from donors already

     8    participating in our apheresis program that were used

     9    to taking dexamethasone.  So I could compare in one

    10    study subject the response to G-CSF dexa stimulated

    11    product versus dexa alone, and there is a marked and

    12    significant difference as you would expect.  This

    13    happened to be the patient that had the highest

    14    increment of all of our patients to granulocyte

    15    transfusions.  This is the increment, not the absolute

    16    count.  It was 6,500 one hour post-transfusion, rose

    17    to 8,400 four hours post-transfusion, and dropped to

    18    a high 2,000 level 8 hours later and at 24 hours was

    19    still significantly above her baseline of almost zero

    20    as opposed to the response to dexamethasone, where it

    21    was a little bit above 1,000 at one hour, and you can

    22    see the trend down here.  

    23                Alloimmunization, as has been stated, is

    24    a major risk of granulocyte transfusions.  In previous

    25    publications, the instance of transfusion reactions is

                                                                        114

     1    90 percent in individuals getting granulocytes who

     2    have preexisting alloimmunization and 11 percent of

     3    those who do not.  In very elegant studies done almost

     4    20 years ago by Jan Dutcher and Charlie Schiffer where

     5    they radio-labeled allogeneic white cells with indium

     6    111 and transfused them, they saw increased pulmonary

     7    retention of cells and decreased trafficking to sites

     8    of infection in individuals who had preexisting HLA

     9    alloantibodies.  20 of 20 successfully migrated to

    10    sites of infection without HLA alloimmunization versus

    11    3 of 14. 

    12                We looked at the NIH retrospectively at

    13    CGD recipients of multiple courses of granulocyte

    14    transfusion therapy in the era where we used

    15    dexamethasone alone mobilized products.  And of 18

    16    patients that we looked at, 14 of 18 had developed HLA

    17    alloantibodies during the course of these

    18    transfusions.  So in some populations, and I suspect

    19    that would be CGD and also aplastics, the instance of

    20    alloimmunization is extremely high, on the order of 80

    21    percent.  Whereas in bone marrow transplant

    22    recipients, it is probably much less because of the

    23    state of suppression of their immune system.

    24                We looked more carefully at patients with

    25    CDG that we transfused.  This is the first six that

                                                                        115

     1    received G-CSF mobilized product.  One with

     2    Aspergillus bacterial pneumonia, diffuse nocardia, an

     3    unspecified fungal pneumonia, bacterial pneumonia, and

     4    staph hepatic abscesses.  Our protocol said we would

     5    not give these cells to patients with preexistent HLA

     6    alloimmunization, but the clinicians taking care of

     7    these patients were so impressed with the response to

     8    granulocyte transfusions in patients without HLA

     9    alloantibodies that they prevailed upon us to make a

    10    deviation to our standard operating procedure.  So in

    11    three of these patients, there were preexistent HLA

    12    alloantibodies and in one there was prior

    13    alloimmunization, but it was not initially evident on

    14    the screen although it became evident later. 

    15    Interestingly, not all patients with alloimmunization

    16    had pulmonary adverse reactions.  This could be

    17    pulmonary infiltrates, fever, chills, dyspnea, or 02

    18    desaturation.  None of the patients without

    19    alloantibodies had reactions.  Three of the patients

    20    with alloantibodies did and one did not.  02

    21    desaturation was seen in two of the four with

    22    allosensitization and was not seen in the other two. 

    23    So it wasn't uniform here either.  An indium labeled

    24    allogeneic white cell trafficking scan was done in the

    25    individual with diffuse nocardia.  She had

                                                                        116

     1    disseminated skin lesions which should have been easy

     2    to see on the scan and all she had was pulmonary

     3    retention with no traffic.  So in this patient with

     4    alloimmunization, again the cells did not go to sites

     5    of infection.  

     6                Very nicely with the help of Dr. Fleischer

     7    and Dr. Malik here at the NIH, a flow cytometric study

     8    using a DHR dihydrorodamine stain was done. 

     9    Dihydrorodamine fluoresces inside neutrophils when the

    10    cells undergo a respiratory burst, which is detected

    11    by the flow cytometer.  Such patients with CGD don't

    12    have granulocytes that can undergo respiratory bursts

    13    and the DHR is zero percent of cells in the wild type

    14    state.  Following transfusion, the percent of cells

    15    that are phagocyte oxidase positive rises to anywhere

    16    from 6 to 64 percent, starts at 0, and was always

    17    greater than 1 percent in all individuals who did not

    18    have HLA alloimmunization.  It was less than 1 percent

    19    -- substantially less -- and this may be some

    20    background activity or background noise, in patients

    21    who did have alloimmunization.  This high of 54

    22    percent was the first transfusion given to this

    23    individual before the HLA alloantibodies became

    24    evident in this serum.  This number actually decreased

    25    to less than 1 percent after the alloimmunization

                                                                        117

     1    occurred.

     2                I would like to end, as Liana has asked us

     3    to, with considerations for the focus of future

     4    research studies.  I have decided these foci into

     5    donor collection methods, components, and recipient

     6    considerations.  As referred to numerous times both

     7    yesterday and today, we need continued long-term

     8    follow-up of healthy recipients of G-CSF to see if

     9    there are any long-term complications.  There do not

    10    appear to be with over 1,000 or several thousand

    11    individuals receiving not only a single dose but five

    12    consecutive daily doses of G-CSF.  The other

    13    consideration is how often we can do this in a

    14    volunteer donor for granulocytes and not for stem

    15    cells.  FDA allows us to do cytopheresis 24 times a

    16    year in a normal donor.  Our policy at the NIH

    17    transfusion medicine department is to allow donors on

    18    this G-CSF mobilized donation protocol to donate no

    19    more often than monthly, but that is an arbitrary

    20    restriction.  Should there be a difference -- should

    21    there be a different restriction for healthy donors

    22    getting G-CSF mobilized products than for routine

    23    cytopheresis donors?  

    24                Collection methods -- certainly with the

    25    CS-3000, enhancements and modifications are needed to

                                                                        118

     1    the apheresis devices to increase the efficiency of

     2    collection and those studies are in progress. 

     3    Components, as we just heard from Dr. Ambruso, it is

     4    very difficult if not impossible for most centers to

     5    collect, test, and transfuse on the same day or even

     6    to collect, test, and transfuse within 24 hours.  So

     7    evaluation of storage solutions and conditions if

     8    ongoing in several institutions is critical to allow

     9    this kind of component to be made available at

    10    multiple blood centers.  

    11                It appears that we have defined a

    12    component.  The component definition is becoming very

    13    crisp.  What we found in a retrospective analysis was

    14    that 75 percent of our components contain greater than

    15    5 times 1010 granulocytes and 90 percent contain

    16    greater than 4 times 1010 granulocytes in an analysis

    17    of about 200 G+ dexa mobilized components.  So you can

    18    define a minimum number.  You can define the apheresis

    19    procedures and the donor preparative regimens to yield

    20    that product.  So the product definition again is not

    21    so much a problem.  What is a problem is determining

    22    efficacy.

    23                So studies of recipients -- and we have

    24    heard this before from other individuals -- a multi-

    25    center, randomized, prospectively controlled study,

                                                                        119

     1    and I think that this will be so expensive and complex

     2    that there probably won't be more than one such study

     3    which is being organized out of the infectious disease

     4    department at the Fred Hutch, or at least there are

     5    discussions of it right now, to identify patients most

     6    likely to obtain survival benefit.

     7                Eligibility criteria, as we have heard,

     8    are deep-seated filamentous fungal infections in the

     9    hematopoietic transplant setting, perhaps in patients

    10    with severe aplastic anemia, and also life-threatening

    11    bacterial infections where we assess the patient as

    12    having a greater than 70 or 80 or 90 percent chance of 

    13    mortality with best available antimicrobial therapy,

    14    and in a separate population of patients who are not

    15    neutropenic, CDG patients with fungal or bacterial

    16    infections.

    17                In terms of assessment of efficacy, we

    18    have designed such a trial for patients with aplastic

    19    anemia in this institution, which is a referral center

    20    for SAA.  Our statistician reviewing our trial told us

    21    that our primary outcome should not be resolution of

    22    infection but should be survival at three months when

    23    she looked at the data we had on patient outcomes in

    24    the first 14 patients treated, and that is different

    25    than what we heard this morning from Seattle where

                                                                        120

     1    resolution of infection was a primary outcome.  You

     2    don't judge platelet transfusions or red cell

     3    transfusions by whether the patient leaves the

     4    hospital.  So this would be sort of a new paradigm. 

     5    And I am not sure which one of these is best, and I

     6    think the statisticians, whether it is hospital

     7    discharge or long term survival at three and six

     8    months or resolution of infection -- the statisticians

     9    should have more discussion on what is the appropriate

    10    primary endpoint.  The other endpoints can be

    11    evaluated in a logistic regression.  But you have to

    12    define the primary endpoint more clearly.

    13                I would like to stop there with the

    14    exception of my last and of course the most important

    15    slide, my acknowledgements to Mr. Jaime Oblitas,

    16    Virginia Morgan, and Sandy Bangham and the outstanding

    17    staff of the NIH Apheresis Center for allowing these

    18    studies to take place.

    19                CHAIRPERSON HARVATH:  Would the speakers

    20    from this morning's two sessions please assemble and

    21    we will give the audience time to put questions

    22    together. 

    23                DR. ADKINS:  Adkins from St. Louis.  As a

    24    patient, if I had a less than 10 or 20 percent chance

    25    of surviving from a progressive infection while

                                                                        121

     1    neutropenic, I guess I would have a problem being

     2    asked to be randomized to receive or not receive a

     3    granulocyte transfusion in a study such as this.  So

     4    I guess I would ask the response of the speakers how

     5    they would feel as a physician who is trying to

     6    counsel patients for a proposed randomized trial of

     7    therapeutic granulocyte transfusions.  If they feel

     8    that is appropriate or how they would go about trying

     9    to convince people to participate on a trial like

    10    this.

    11                DR. LEITMAN:  That is certainly one of the

    12    most difficult questions.  Our aplastic anemia trial

    13    is not yet running.  We have had in the last two

    14    months several patients with deep-seated filamentous

    15    fungal infections who we knew the mortality would be

    16    100 percent with conventional therapy.  The attendings

    17    that month were asked whether they would have

    18    randomized those patients if the trial were active and

    19    they said, oh, I don't think so.  But then when we

    20    look at our outcome data, all such patients have died

    21    with granulocyte transfusions, 3 out of 3.  Only the

    22    bacterial infection patient survived.  All three had

    23    improvement in their infections over the first two to

    24    three weeks.  But as one waits that six weeks or eight

    25    or twelve to recovery of their own counts, it becomes

                                                                        122

     1    increasingly difficult to support with granulocyte

     2    transfusions and then alloimmunization is likely to

     3    occur as well.  So given the data, though small

     4    numbers of patients here and at other sites, I think

     5    that that is the way to do the study, to randomize.

     6                DR. ADKINS:  I think each of you shared

     7    your enthusiasm for this area and actually have been

     8    very positive about the outcomes you have observed. 

     9    I am all for randomized trials to try to prove whether

    10    or not these things work.  I guess I am questioning is

    11    this the right setting as a therapeutic maneuver.  In

    12    my own talk I will discuss kind of a strategy we have

    13    taken as kind of a prophylactic maneuver.  I think

    14    that that is another way to look at efficacy analyses

    15    with granulocyte transfusions, and perhaps a more

    16    acceptable way from a patient perspective.  So, again,

    17    if all of us, let's say, were going to join up and do

    18    a Phase III trial, I think it is very important that

    19    we are all convinced that we can comfortably go to a

    20    patient and say these are your two options.  I don't

    21    know which one of these provides the best benefit and

    22    I am very comfortable in recommending you to receive

    23    or not receive by flip of the coin a granulocyte

    24    transfusion.  So I just would encourage us all to

    25    think about this as we leave today.  

                                                                        123

     1                DR. PRICE:  Well, I might comment.  I

     2    mean, I agree with you that it is going to be a

     3    problem.  I personally -- I don't think I would have

     4    a problem randomizing.  I don't know whether this

     5    stuff works or not.  But the more we talk about this

     6    too, the more there are going to be enough people

     7    around who are going to have a problem with that

     8    randomization, and I think we are hearing about that

     9    now and seeing it.  One of the possibilities that we

    10    have at least batted around a little bit would be

    11    whether in a randomized trial there could be an out

    12    and some sort of a deal that says if you are in the

    13    control arm and you start going down the tubes that

    14    there is an escape clause and you can switch over.  So

    15    that is another possibility.

    16                One of the problems that we have faced in

    17    terms of considering a prophylactic trial is that if

    18    you look at the fraction of the patients that get

    19    these kinds of infections -- I mean, maybe it is 10

    20    percent of the patients or 5 or 10 percent of the

    21    patients that get one of these kind of infections that

    22    we end up treating, that we are going to have to be

    23    giving 10 or 20 people granulocyte transfusions for

    24    the one we would have given therapeutically.  And when

    25    you start looking at how much that is going to cost

                                                                        124

     1    and from a blood center point of view how many

     2    granulocyte donors we are going to have to find every

     3    day, that is another real problem that comes into it

     4    when you are considering the prophylactic.

     5                DR. ADKINS:  Maybe I should just stay up

     6    here.  But I guess, Tom, if this is historically what

     7    has happened in the past with granulocyte

     8    transfusions, if I am randomized to not receive

     9    granulocytes and I have got a fungal infection and it

    10    gets worse after that randomization, the tendency is

    11    going to be to take care of that patient and to then

    12    go on to later give them those granulocytes "off

    13    study".  And then if your analysis is to determine

    14    survival from a statistical standpoint, you are never

    15    going to be able to prove that if you allow people to

    16    "be rescued" for want of a better word.  So this is a

    17    very challenging area to try to prove efficacy if you

    18    focus this as a therapeutic maneuver.  So, again, I

    19    think you really have to think about this very

    20    carefully based on the historical trials that have

    21    been done with randomized trials.  We know that these

    22    are practical problems that we are going to run into,

    23    and how are we going to deal with them.  How are we

    24    going to manage then and how are we going to account

    25    for that statistically?  That is not going to be an

                                                                        125

     1    easy -- there is no easy answer to that and there may

     2    be no answer to that, which is I think the point I am

     3    trying to make.

     4                DR. PRICE:  I agree.  

     5                DR. DALE:  I'd just make a brief comment. 

     6    In the randomized trials that Ron Strauss reviewed,

     7    the survival with transfusions was roughly 11 percent

     8    for invasive molds.  So historically, there is really

     9    not much evidence.  You would have to base evidence of

    10    treatment benefit on new results like were described

    11    today.  And they are not certainly clear cut at this

    12    point to know the benefits.  So I feel like from an

    13    ethical standpoint, it is a reasonable thing to do,

    14    particularly before we and others encourage the more

    15    widespread application of this very expensive and

    16    resource consumptive technology.

    17                DR. TORLINI:  Hi, Sergio Torlini Inova

    18    Fairfax Hospital.  Since the granulocytes go to a

    19    population that is almost 100 percent immune

    20    suppressed and therefore at risk of graft versus host

    21    disease and therefore the products have to be

    22    radiated, I would like to know if anybody has any data

    23    as far as the radiation on granulocyte function, I

    24    mean anything recent, and also on the dose of

    25    radiation on that.

                                                                        126

     1                DR. DALE:  I will comment briefly.  We

     2    haven't studied that in Seattle.  At MD Anderson,

     3    there are some recent studies that do suggest some

     4    modest degree of cell injury with radiation.  That

     5    may, in fact, be overcome by the addition of other

     6    cytokines, the cytokine being gamma interferon, for

     7    example, having a protective effect.  It is a murky

     8    area, though.  And basically I think what has been

     9    done universally is to irradiate cells assuming they

    10    are not damaged.  So it is something that needs to be

    11    better studied.

    12                DR. LEITMAN:  There is a very nice study

    13    by Kasberg, Lur, and colleagues in Switzerland

    14    published in Blood in 1993 where I can't remember the

    15    dose, it was 2,500 or 3,000 centigrade, were given to

    16    the product and before and after radiation very

    17    careful studies of migration and chemotaxis and

    18    fungicidal and on the respiratory burst activity. 

    19    There were 5 or 6 parameters looked at and there was

    20    no difference pre and post to radiation.  

    21                DR. DIAZ:  Jose-Luis Diaz from Mitre

    22    Pharmaceuticals in La Jolla.  I have a simple nuts and

    23    bolts question for Dr. Ambruso, which is how did you

    24    measure apoptosis in the cells that you were

    25    measuring?

                                                                        127

     1                DR. AMBRUSO:  How did we measure it?

     2                DR. DIAZ:  Yes.

     3                DR. AMBRUSO:  We looked at morphologic

     4    criteria.  That slide that I showed you showed a

     5    distinct change in the morphologic appearance of the

     6    nuclei.  And we looked at percent of live apototic   

     7    cells.

     8                DR. DIAZ:  Right. And the other thing was

     9    in some of the other functional assays, how did you

    10    distinguish the response you were getting -- how did

    11    you determine that the response you were getting was

    12    from a neutrophil and not from something like a

    13    monocyte, for example.  Obviously monocytes can burst,

    14    et cetera.

    15                DR. AMBRUSO:  Sure.  Our preparations are

    16    95 to 98 percent neutrophils, and that was checked

    17    each time that the cells were separated.

    18                DR. DIAZ:  Oh, I see.  So you took the

    19    samples and then did a separation?

    20                DR. AMBRUSO:  Right.  There was a further

    21    separation.

    22                DR. DIAZ:  Okay.  Thanks so much.

    23                AUDIENCE MEMBER:  I would like to comment

    24    on the respiratory distress that you saw, Susan, with

    25    some of your granulocyte preparations.  We see a lot

                                                                        128

     1    of respiratory distress after transfusion and only the

     2    anti HLA2 agglutinating antibodies of the HLA

     3    antibodies apparently cause respiratory distress. Now

     4    the antigranulocyte antibodies for the granulocyte

     5    antigen specifically also cause respiratory distress,

     6    and we find that we have many, many more

     7    antigranulocyte antibodies causing respiratory

     8    distress than HLAs.  But in HLA territory, if you can

     9    avoid giving anti-HLA2's -- giving HLA2's to the

    10    people who might have anti-HLA2's, you might avoid

    11    that small group.  But they look to be only about a

    12    fifth or a tenth of the respiratory distress cases

    13    that we have before us.  So I wonder if anybody else

    14    has any different experience with that.  We have been

    15    very interested in that because it is a very serious

    16    complication.  

    17                DR. LEITMAN:  In the retrospective

    18    analysis of pulmonary complications in CDG recipients

    19    of granulocytes that was published by Dave Stroncek a

    20    year ago with the CDG patients here, the pulmonary

    21    reactions were seen with HLA alloantibodies alone in

    22    the absence of anti-neutrophil antibodies, and all

    23    patients were screened for presence of both anti-

    24    neutrophil and anti-HLA.  I am almost certain that it

    25    wasn't only HLA A2.  Most of the recipients had multi-

                                                                        129

     1    specific antibody screens.  I am not sure we can pull

     2    out the A2.  But I don't think it was only HLA A2.  

     3                DR. PRICE:  Becky, are you referring to

     4    granulocyte things only or is this trolley type stuff

     5    you are talking about?

     6                AUDIENCE MEMBER:  This is trolley type

     7    stuff.  Because we have seen it with the anti-

     8    granulocyte.  We have seen it anti-HLA2, but with

     9    other kinds of anti-HLA.  I would certainly be willing

    10    to learn.  I need to go read David's paper again

    11    obviously.

    12                DR. AMBRUSO:  Pulmonary reactions related

    13    to transfusions is something that our laboratory has

    14    been interested in in the past few years and it

    15    certainly occurs for reasons other than antibodies. 

    16    And these patients, aside from the granulocyte issue,

    17    certainly are receiving other products and have other

    18    illness-related problems that certainly could

    19    predispose them to that.  I am surprised that, in

    20    fact, there aren't more and it is something, more

    21    reactions, and may not necessarily be related to the

    22    antibodies or the granulocytes themselves.  But there

    23    are other compounds that have been indicated --

    24    cytokines, lipids that were intensely interested and

    25    that certainly have an etiologic role.  So this gets

                                                                        130

     1    very murky when we start talking about pulmonary

     2    reactions and there are lots of ways that it can

     3    happen.

     4                DR. KLEIN:  Harvey Klein, NIH.  Is anyone

     5    looking at or concerned with the issue of CMV

     6    transmission when patients who are immunosupressed get

     7    these large numbers of granulocytes over long periods

     8    of time?

     9                DR. PRICE:  Well, I mean the other people

    10    can answer this.  Our routine, of course, is for any

    11    recipient who is CMV negative to provide donors that

    12    are CMV negative.  Whether a CMV positive donor

    13    getting CMV positive stuff is going to get some other

    14    strain and do him some damage is not something that we

    15    have addressed.

    16                DR. AMBRUSO:  We have routinely used the

    17    same or adopted the same approach and that is either

    18    using sero negative or using leuko-reduced.  But most

    19    of what we are doing still is sero negative -- CMP

    20    sero negative for patients that would fall into that

    21    category.  And I assume we would continue.

    22                AUDIENCE MEMBER:  Could I ask anybody,

    23    what would be the upper level of neutrophils that you

    24    would exclude a person from this proposed trial?

    25                DR. DALE:  Do you mean what -- 

                                                                        131

     1                AUDIENCE MEMBER:   Yes, what degree of

     2    neutropenia do you think that you would think

     3    neutrophil transfusions would have an impact?

     4                DR. DALE:  That is a good question.  The

     5    traditional or historic level of 500 is a level that

     6    is cut off as a pretty high level.  The risk of

     7    infection is the severity of infection is considerably

     8    more if you use 200.  You could argue for using even

     9    a higher threshold based upon studies of functional

    10    deficiencies of the neutrophils produced after

    11    transplantation in the early phases of hematopoietic 

    12    recovery.  I don't think that any choice would be less

    13    than arbitrary, though.  It would fall somewhere

    14    probably between 200 and 1,000.

    15                AUDIENCE MEMBER:  Could I just ask another

    16    question?  If you were treating a fungal infection,

    17    would you disqualify people for this trial on

    18    steroids?

    19                DR. DALE:  That is another very good

    20    question.  In the post-transplant period when people

    21    are on steroids, that is going to be an important

    22    factor in their infection.  And it has been thought

    23    that it would be useful to transfuse people with

    24    higher counts because again the endogenous cells are

    25    perhaps ineffective.  That is mostly speculation,

                                                                        132

     1    though.  I don't think anyone -- there is not a right

     2    answer.  So that whatever trial were conducted would

     3    be some consensus.  But certainly people I have talked

     4    with have suggested that for fungal infections after

     5    transplant for people on steroids, you should consider

     6    people with higher counts than 200 or 500.

     7                AUDIENCE MEMBER:  But wouldn't they

     8    suffer, then, the same paralysis that is going on in

     9    vivo?

    10                DR. DALE:  Yes and no, and that depends

    11    upon whether the effects of steroids or

    12    immunosuppressive drugs have their effects on

    13    developing cells or developed transfused cells.  And

    14    I suspect it is the former, not the latter.  But,

    15    again, another good research question.

    16                DR. STRONCEK:  Dave Stroncek, NIH.  A

    17    couple of comments.  One is I know the average data

    18    looks very good for the increments and neutrophil

    19    counts after transfusions and patient outcomes.  But

    20    I have seem some of these same patients anecdotally

    21    and I don't think the average data -- I don't think

    22    the whole picture is quite as rosy as the average data

    23    tends to show.  That said, I think when you consider

    24    clinical trials, I have a similar concern about the

    25    patients studied as Dr. Adkins but for a different

                                                                        133

     1    reason.  I think bacterial infections granulocyte

     2    transfusions will work great for.  But the number of

     3    patients we see for that have bacterial infections not

     4    responding to antibiotics is really very rare.  The

     5    biggest problem seems to be patients with fungal

     6    infections.  And I have a concern that the granulocyte

     7    transfusions may be effective in increasing white

     8    count, but they still might not be effective in

     9    resolving fungal infections, or at least not effective

    10    enough to see in a reasonable trial.  So for that

    11    reason, if you set up a trial that just looks at

    12    treating fungal infections in neutropenic patients, it

    13    may fail.  So it may be worthwhile to try it in a

    14    different patient population, maybe in the

    15    prophylactic setting. 

    16                And the other comment too is I know in the

    17    past we had to look at really patient survival as an

    18    outcome because that was the only outcome measure we

    19    had.  You couldn't measure increase in granulocyte

    20    counts. But I think that needs to be relooked at in

    21    further studies.  A number of the studies on cytokines

    22    that have been given to patients with chronic

    23    neutropenia and neutropenic patients getting

    24    chemotherapy have shown shorter days of neutropenia

    25    and shorter days of hospital stay and decreased

                                                                        134

     1    febrile incidence, but I don't think they have been

     2    held to the same standard to show that there is less

     3    infection -- less fungal and bacterial infection.  So

     4    I just caution that we don't set ourselves up to fail

     5    because we are being more strict with the criteria to

     6    say granulocyte transfusions don't work.  If you do it

     7    because we are more strict than any other standard we

     8    hold for other blood products or other drugs.  

     9                DR. HENDERSON:  I am Theresa Henderson

    10    from Georgetown University and I have a couple of nuts

    11    and bolts questions for Dr. Ambruso.  I just wanted to

    12    clarify that the cells that you were testing were not

    13    the apheresed product but had been stored and cleaned

    14    up by fical or something like that?

    15                DR. AMBRUSO:  Right.  The actual data that

    16    I have showed was cells from the -- they were drawn

    17    from the patient before any G-CSF had been given.  And

    18    approximately one  hour after the dose of G-CSF on day

    19    4.  So that is the fifth dose of G-CSF.  Subsequent to

    20    that, the patients underwent granulocyte collections

    21    and I didn't show the data but put it in a summary

    22    slide that we looked at additional studies in those

    23    granulocytes.  And we really didn't find much

    24    difference in terms of the function for the first 24

    25    hours.  I had 24 hours of storage.

                                                                        135

     1                DR. HENDERSON:  Oh, that is good to know. 

     2    And you said that you looked at C-11B and saw no

     3    significant changes.  I wondered if you looked at

     4    Selectin-62?

     5                DR. AMBRUSO:  We did not.

     6                DR. HENDERSON:  And you also touched on

     7    the cytosolic calcium increase.  I am sorry, I don't

     8    understand -- I am new at this, so I don't really

     9    understand the significance of looking at that.  Could

    10    you enlighten me?

    11                DR. AMBRUSO:  We did that in part because

    12    we wanted to look at a biochemical parameter that we

    13    thought was related.  I mean initially we did it and

    14    were excited related to the chemotaxis.  But obviously

    15    calcium and signaling related to calcium is something

    16    that is significant to all functions of the

    17    neutrophil.  We also did it in response to some

    18    studies in another patient group.  There is a group of

    19    patients who have neutropenia and neutrophil

    20    dysfunction that are associated with glycogenesis 1B. 

    21    These individuals, when you put them on G-CSF, their

    22    neutrophil counts are restored.  They are not

    23    neutropenic.  They still have a chemotactic defect and

    24    their cytosolic response to calcium, which was

    25    aberrant to begin with without treatment with G-CSF

                                                                        136

     1    normalized.  So we wanted to look at a group of

     2    control individuals given G-CSF to see if that affect

     3    was consistent.  It is consistent.  I don't know if it

     4    has any relationship to the defects or anything that

     5    was shown in the function of the cells in normal

     6    patients given G-CSF.

     7                DR. HENDERSON:  Thank you.

     8                DR. DIAZ:  Just a quick point.  About L-

     9    Selectin.  We did some studies on isolated neutrophils

    10    looking at 62L, which is marked for L-Selectin, and

    11    what we found was that in isolating neutrophils, the

    12    L-Selectin dies off slowly and after about 24 hours,

    13    there is only about 50 percent.  And then by about 48

    14    hours, there is only about 20 percent left.  If at any

    15    of these points you actually activate the cell by

    16    giving it a stimulus, it disappears within an hour or

    17    very quickly.  

    18                DR. LEITMAN:  I have a question for the

    19    FDA.  You can now define a product much better than

    20    you could before.  You can now define a measurable

    21    outcome in a transfusion recipient.  You can even set

    22    criteria for assessing that outcome, an increment in

    23    the granulocyte count at certain times that is

    24    sustained for a certain length of time.  So your

    25    definition of the component of how you treat the donor

                                                                        137

     1    and what you measure immediately after transfusion in

     2    the recipient is quite well defined.  Would the FDA,

     3    in considering licensure of this product, have to see,

     4    want to see, insist on seeing the results of a

     5    randomized clinical trial?  And what would be the

     6    endpoints that they would look at of that trial?

     7                CHAIRPERSON HARVATH:  Susan always asks me

     8    these tough questions.  That is why we are holding the

     9    conference.  We wanted to hear from you what you feel

    10    is an appropriate approach because we have heard that

    11    this is very expensive.  We also hear that normal

    12    donors may be asked to be receiving cytokine

    13    repetitively.  And as you know, we have to constantly

    14    look at risk benefit in terms of public health issues. 

    15    I agree with everything you have said.  I think that

    16    you have made enormous strides in defining a component

    17    and in terms of the cell biology.  I think that that

    18    is the easier part of all of this.  One of the things

    19    we wanted to hear from this workshop and yesterday

    20    were your concerns about the exposure of normals and

    21    the effects long term.  So I don't really have an

    22    answer for your question, Susan.  I think what we

    23    wanted to do was hear what you were all comfortable

    24    doing.  We know some folks have talked about

    25    collectively doing a randomized trial.  We know the

                                                                        138

     1    NIH is interested in hearing what the interest is in

     2    that and in determining whether there would be support

     3    to try and do that.  We also know that is going to be

     4    very expensive to be done and I think you said it very

     5    accurately this morning that it probably would be done

     6    once and hopefully done so that one gets a clearer

     7    answer.  

     8                So I honestly don't have an answer.  I

     9    can't speak on behalf of the Agency.  As you know, I

    10    am interested in the cell biology and also I think we

    11    have an obligation to ask investigators who are

    12    collecting these products how comfortable they are

    13    with giving their normal pedigree blood donors

    14    cytokines and giving them cytokines perhaps on more

    15    than one occasion.

    16                One question I have for all of you along

    17    that line -- and I am sorry I don't answer your

    18    question but that is my non-answer.  One question I

    19    have for you is I have heard all of you say that you

    20    are working with your pedigree donors.  So you really

    21    have a very clear picture of their medical histories

    22    and their hematologic picture.  When you do a complete

    23    blood count on someone, are you including a

    24    differential in that and how would you feel about

    25    giving G-CSF or some other cytokine to someone where

                                                                        139

     1    you only had a blood count and not necessarily were

     2    resting assured that they had a normal differential? 

     3    Do any of you  have concerns that there might be an

     4    individual perhaps with perhaps a pre-leukemic state

     5    that could possibly be missed?  That might not be your

     6    normal pedigree donor population that you have

     7    followed over the years and have a clear health

     8    picture on.  But something we have kind of wondered

     9    about and we don't have the answer to it is what kind

    10    of -- would you include a differential in your workup

    11    initially in a new donor situation?  And that is for

    12    everyone.

    13                DR. PRICE:  Well, we do a differential,

    14    but by the time we get the answer back, the G has

    15    already been given to the donor.  Like you say, these

    16    are donors we know.  But a guy who is going to develop

    17    leukemia will do it at point X and that may happen. 

    18    I think -- I mean, my read on this is that there have

    19    been a lot of people looking at this and the evidence

    20    of giving one shot of G even to somebody who is

    21    developing leukemia that it is going to do him any

    22    harm would be about zero.

    23                DR. AMBRUSO:  We, like you, are doing

    24    counts with automated differentials on our granulocyte

    25    donors as well as we are doing counts on platelet

                                                                        140

     1    donors.  That is something that we worry about and I

     2    wonder if you would be more concerned or more likely

     3    to run into problems with very high counts -- just

     4    with the white count and looking at that carefully

     5    than with the differential.  I am not sure that we

     6    collect a lot of reasonable data.  The parameter that

     7    should be different is looking at counts that exceed

     8    or are above or below certain levels in terms of the

     9    risk for leukemia or other problems.

    10                DR. LEITMAN:  We automatically do a CBC

    11    with an automated differential on all our donors

    12    including platelet donors, and part of the reason is

    13    to look at their platelet counts to qualify them for

    14    the next platelet pheresis donation.  Because the

    15    granulocyte donor one month later is most likely to be

    16    donating platelets and not granulocytes again.  And

    17    that is the reason I think most centers if not all

    18    have to do a pre-pheresis count.  Like Tom, our count

    19    is obtained the day of pheresis in the current

    20    iteration of the study so that we get the count after

    21    they have been given the stimulant.  Does the count

    22    have to include a differential?  It is almost a moot

    23    point since the automated instruments provide the

    24    differential.  But I don't see in the absence of a

    25    trial the critical necessity for a differential.

                                                                        141

     1                DR. DALE:  I would just comment too that

     2    this safety issue, that is, a single dose must be an

     3    extremely small risk.  Because the substance that is

     4    used as a drug is really very close to the natural

     5    hormone and a surge of this magnitude probably happens

     6    many times in a person's lifetime.

     7                AUDIENCE MEMBER:  I have a question.  What

     8    are the legalities about giving G-CSF to an unrelated

     9    donor?  I know that in our institution we would have

    10    to go through IRB to get this approved.  Is that

    11    pretty much the way it stands for everybody right now?

    12                DR. DALE:  We have -- we do it with IRB

    13    approval and informed consent.  

    14                DR. LEITMAN:   I think in this

    15    institution, all G-CSF given to normal donors on

    16    numerous protocols, not only ours, is done with IRB

    17    informed consent.

    18                AUDIENCE MEMBER:  All right.  And I guess

    19    a follow-up question is what exactly -- how do you

    20    modify your informed consent form for a G-CSF?  I

    21    mean, how extensive is it?  Several pages?  Just a

    22    paragraph?  

    23                DR. LEITMAN:  It is four pages.

    24                AUDIENCE MEMBER:  A paragraph.  Thank you. 

    25    We were thinking of producing a movie to go with it to

                                                                        142

     1    show.

     2                DR. PRICE:  And I think that although

     3    maybe everybody up here does it in IRB, I think there

     4    are places around who don't do it in IRB.  I still

     5    think they have a consent form and explain to the

     6    donor what is going on.  But I figure that there is

     7    enough information around that as long as they are

     8    keeping track of things that it is not really a

     9    research thing.  I think that is not undefensible. 

    10    Did I say that right?  Too many negatives?

    11                AUDIENCE MEMBER:  Having an almost 20-year

    12    history in apheresis collections and a nursing

    13    background, I have to preface this by saying that I am

    14    a donor advocate and having been a nurse and being

    15    trained to try and help people get better, I have a

    16    real internal conflict going on with asking volunteer

    17    donors to take a drug that we don't have long-term

    18    studies as to show what happens with these donors with

    19    exposure to the drug.  And then tied in with that is

    20    the fact that there really -- if I am hearing

    21    correctly what I have heard today, the efficacy of

    22    what we are trying to collect really has not been

    23    established.  And I am real worried and terribly

    24    protective of the donors and what we ask of them.  I

    25    think also as a personal observation of donors that we

                                                                        143

     1    have worked with in the past, yes, the donors will

     2    come back after many circumstances and I would be

     3    willing to wager that because they are a captive

     4    audience, they are a very dedicated conscientious

     5    group of people that would probably -- and I

     6    acknowledge that I have no data to support this fact

     7    other than a gut feeling -- but I think the donors

     8    would probably come back no matter what we asked of

     9    them, and where do we draw the line between what we

    10    ask of our donors and what we are trying to accomplish

    11    in our patients?

    12                DR. DALE:  I would just comment.  I think

    13    that is a very good statement and in fact provides

    14    part of the rationale for a randomized trial.  It is

    15    not to do something with donors which is not

    16    convincingly proven to be of value.  Protect their

    17    interest as well.  On the other hand, I think being

    18    open with people and honest about the potential

    19    benefit and the known risk that the donor population

    20    can make an informed choice.  As adults, some will say

    21    yes and some will say no and that is okay.

    22                DR. AMBRUSO:  I think you bring up an

    23    important point and I think we really do need to know

    24    long term effects.  I guess I am not concerned about

    25    what happens with one dose of G-CSF and then the donor

                                                                        144

     1    goes merrily on their way.  People who are involved in

     2    providing blood products know that you are relying on

     3    the bone marrows of a very few individuals to support

     4    the rest of the community.  And it is really not a

     5    single donation per year that we are concerned about. 

     6    It is the donation of something of someone who might

     7    undergo this procedure many times in a year and that

     8    is completely undefined.  As you were saying this, I

     9    am reminded also that we have many, many dedicated

    10    platelet donors.  And you know, we don't know long-

    11    term what happens to somebody who is having their

    12    platelets collected many, many times a year -- 23 or

    13    26 times a year.  You know, we don't have long term

    14    data on that, but we still go ahead and do those times

    15    of collection.  So this is a muddy area, but I think

    16    we really do -- with G-CSF mobilization, we do need to

    17    get some long-term data, particularly on the donors

    18    who are going to be dedicated and are going to donate

    19    more than once a year.  If they are going to be

    20    denoting once a month, I think there is some long term

    21    information that we need on them.

    22                DR. LEITMAN:  The NIH has been collecting

    23    granulocytes for transfusion for greater than 15

    24    years, maybe for 20 years. Since the Michler studies

    25    in the 1970's about starch and steroid to optimize

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     1    collection efficiency, donors have routinely been

     2    given dexamethasone plus starch.  Neither of those

     3    medications are licensed for use in obtaining

     4    granulocytes because granulocytes aren't a licensed

     5    product.  So the past two decades in this field has

     6    been characterized by giving donors drugs that do have

     7    adverse effects.  45 percent of donors getting dexa

     8    don't have a good night sleep.  They have nightmares

     9    and insomnia.  I see nothing in the past decade of a

    10    history of administering G-CSF to normal, healthy

    11    individuals that increases my concern that G-CSF has

    12    any long term, adverse consequences above and beyond

    13    what we know about dexamethasone and starch.  Acutely,

    14    certainly, it causes more discomfort, which is why I

    15    think you can expect a 10 percent dropout in

    16    individuals participating in this, which is fine.

    17                AUDIENCE MEMBER:  I would hope -- this may

    18    be rather unpopular, but I would hope that the FDA

    19    would like to see or would want a randomized trial, an

    20    appropriate trial.  I think one could ask the question

    21    that if you cannot demonstrate efficacy, number one

    22    does it matter and number two, who do you decide to

    23    give it to?  Are you going to give it to a huge number

    24    of patients who may not need it or who don't need it

    25    or where you can't demonstrate efficacy?  So I think

                                                                        146

     1    to study it and to determine how you ought to use it,

     2    you really need an appropriately designed trial.  It

     3    may be too late to do that and this is a plea,

     4    perhaps, fir people to begin to do appropriately

     5    designed trials very early in the course of the

     6    development of a new component.  Many years ago --

     7    some of us are old enough to be more interested in

     8    history and perhaps unrecorded history.

     9                Everybody knew that fibrinogen given to a

    10    woman bleeding from low fibrinogen after a pregnancy

    11    that the fibrinogen was effective until a major

    12    university did a comparison trial of fibrinogen versus

    13    no fibrinogen and found that the increase in

    14    fibrinogen was as rapid without it as it was with it. 

    15    That was a pretty well defined derivative.  It could

    16    be measured and you gave a dose that you knew about. 

    17    So I think being able to define a component does not

    18    necessarily mean that it is going to be effective and

    19    useful.

    20                DR. SHAPRIO:  Arell Shapiro from Life

    21    Source.  From the data presented, I didn't get a very

    22    good understanding of how the patients are treated. 

    23    Is it -- you know, once it is ordered, is it daily? 

    24    I mean, Dr. Leitman, in your presentation it was daily

    25    except for the weekends.  Is that how -- it is just

                                                                        147

     1    ongoing until the person either recovers back their

     2    white count?  I mean, what are the endpoints and when

     3    do people give up?

     4                DR. LEITMAN:  The call comes to the

     5    consult service to consider granulocyte transfusions

     6    for a patient.  One of our fellows immediately within

     7    hours sees the patient and performs a full evaluation

     8    and it is not a 100 percent approval.  The Transfusion

     9    Medicine Department takes a very active role in

    10    deciding and looking at everything involved with that

    11    patient as to whether this is a good thing to do. 

    12    Once we start, it is daily, omitting in general

    13    weekends but depends on how critical the patient's

    14    status is and we do have the potential to collect on

    15    Saturday and Sunday and holidays, which we do.  

    16                You raise a very good point.  In some

    17    individuals, the increment after the first two

    18    transfusions is so high and so sustained one can ask

    19    whether it is necessary to continue daily transfusions

    20    especially in small kilo recipients such as children

    21    or small sized adults, and in such individuals on such

    22    cases with discussion with the clinical staff we have

    23    moved to every other day transfusions and the

    24    neutrophil count does not diminish generally to zero.

    25                AUDIENCE MEMBER:  So basically you just do

                                                                        148

     1    it by following the patient and see what their

     2    response is.  Have you thought about doubling up on a

     3    Friday so that you could sustain them over the weekend

     4    -- give them a double dose?  

     5                DR. LEITMAN:  The data on storage for

     6    longer than 24 hours is problematic.  So we do it

     7    every day.

     8                AUDIENCE MEMBER:  No, I mean infuse both

     9    on Friday.  

    10                DR. LEITMAN:  Since the increment is in

    11    the 2,000 range with the single transfusion, that is

    12    enough for us.  We have not done double doses.

    13                DR. PRICE:  Our general approach is

    14    similar.  Once we start, we try to do it daily.  We

    15    even do it on the weekends.  The endpoints are a

    16    little bit muddier.  Part of this comes back to the

    17    question that was asked earlier of what is a good

    18    neutrophil count to have.

    19                And once you get it up to 2,000 are you

    20    okay or is somebody with a fungal infection, would

    21    they rather have a count of 8,000 than they would of

    22    3,000?  And our feasibility study didn't really

    23    control that and it was kind of up to the clinicians. 

    24    But that would have to be very carefully laid out for

    25    a real trial.

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     1                AUDIENCE MEMBER:  We have donors who are

     2    willing to take G-CSF for many days in a row.  Have

     3    you looked at seeing if you can collect granulocytes

     4    from your community pools on a daily basis for say

     5    four or five days and would this be of any benefit?

     6                DR. PRICE:  We haven't.  Part of this is

     7    that I -- I mean, I think you are in two different

     8    ball games when you are talking about a one-shot thing

     9    and a multi-thing as Dan was talking about.  I mean,

    10    you run into are the cells the same after five days,

    11    say of G, as they are 12 hours later.  You also run

    12    into issues of cell separation efficiencies if the

    13    cells aren't really different.

    14                But I think at least from my point of

    15    view, that wasn't something I was willing to ask a

    16    regular pheresis donor to do.  We have -- as you know,

    17    Scott, on our early studies we did that with the bone

    18    marrow donors from the Hutch.  And when we did that,

    19    there were several of those donors that did have some

    20    problems getting daily starch.

    21                We had to stop collections because people

    22    got fluid overload and got bad headaches and things

    23    like this.  It didn't happen all the time, but there

    24    was enough of that that for our community donors it

    25    seemed to me that it was sort of above and beyond the

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     1    call of duty just to do it once.

     2                AUDIENCE MEMBER:  That is the reason why

     3    we use pentastarch in our repheresis unit because of

     4    the multiple collections that they will be undergoing

     5    over -- up to 12 days is what we allow at our center.

     6                DR. LEITMAN:   I just want to agree with

     7    Tom Price.  There is a limit beyond which I will not

     8    ask a normal healthy community volunteer to do

     9    something even under the auspices of trial, and I

    10    think one granulocyte aphoresis collection is all I

    11    would ask them to do rather than serial.  We have

    12    occasionally, when we have been unable to get a donor

    13    in over the holidays or whatever, I have had a donor

    14    donate two weeks after their last donation.  But that

    15    is a very rare event, once a month is what I feel

    16    comfortable asking an individual to do.  It is really

    17    an entire day dedicated to apheresis.  They don't feel

    18    that well afterwards.  Their work is affected and

    19    their home life is affected.  I think that is as much

    20    as I would ask.

    21                DR. PRICE:  And another issue that comes

    22    up there, and I don't know if you are still doing

    23    this, Scott -- but when we were doing the family

    24    donors, those guys had central lines and that is also

    25    something I am not too interested in having a regular

                                                                        151

     1    community donor have to have.  

     2                DR. CONFER:  Dennis Confer, National

     3    Marrow Donor Program.  On the issue of G-CSF safety,

     4    I hope it is sort of a rhetorical question, but the

     5    question is, is there really anybody among us who

     6    would give G-CSF to a normal donor if in fact we

     7    thought there was a reasonable chance that it would

     8    cause some late hematologic effect?  If we thought

     9    that there was some reasonable chance that in fact

    10    these donors would develop leukemia at a higher rate

    11    or if we thought that there was some reasonable chance

    12    that in fact these donors might develop aplastic

    13    anemia at old age, I think none of us would give G-CSF

    14    or any other hematopoietic growth factor to a normal

    15    donor.  I think in the same time, I am convinced in

    16    making preparations to give G-CSF to volunteer

    17    hematopoietic stem cell donors, I am confident that

    18    this drug has no long-term effects.  But I think to

    19    demonstrate that, the best way is to collect the long-

    20    term follow-up data.

    21                And we will make plans to follow these

    22    donors for as long as we can.  And it is interesting

    23    because it is something that we talked about

    24    yesterday, that we haven't done it with bone marrow

    25    donors.  That is truly a deficiency.  We also need to

                                                                        152

     1    follow the routine bone marrow donors for as long as

     2    we can to prove that that in fact doesn't cause late

     3    effects, which again we are confident or we feel

     4    reasonably confident that it doesn't.  

     5                But the other thing to keep in mind is

     6    that I can assure you that people who get G-CSF will

     7    develop leukemia.  Because normal people develop

     8    leukemia who have never been exposed to G-CSF.  And

     9    the real question is not how many cases or whether

    10    people develop leukemia, the cases is how many cases

    11    develop and is that different than what would have

    12    been expected among a normal control population.  So

    13    the data we collect really has to be very

    14    comprehensive.

    15                And it has to be compared to an

    16    appropriate control population in order to determine

    17    whether the incidence of leukemia is, in fact,

    18    excessive.  Because it will occur.  And I know from

    19    experience that it has occurred in bone marrow donors,

    20    both before and following bone marrow donation. So it

    21    is a matter of how much, and that is just going to

    22    take a long time to answer that question.

    23                DR. PRICE:  Yes, I think -- because what

    24    we are doing is the mortality of our community donors

    25    eventually is 100 percent.

                                                                        153

     1                CHAIRPERSON HARVATH:  With that concluding

     2    statement, I would like to thank all of you very much

     3    for I think a very informative session this morning. 

     4    We will convene in an hour.

     5                Thank you.

     6                (Whereupon, at 12:05 p.m. the workshop

     7    recessed for lunch to reconvene this same day at 1:10

     8    p.m.)

     9

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                                                                        154

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

     2                                             (1:10 p.m.)

     3                DR. STRONCEK:  I am  Dave Stroncek and I

     4    am going to moderate this session.  We have three

     5    presentations and then after that we will have a panel

     6    discussion.  Following the panel discussion, we only

     7    have three abstracts which will be presented and then

     8    a little further discussion.  So we anticipate we will

     9    probably get done a little early today.  But the first

    10    lecture today will be by Dr. Douglas Adkins.  Dr.

    11    Adkins is Assistant Professor of Medicine at

    12    Washington University School of Medicine, and he is

    13    the medical director of their National Marrow Donor

    14    Transplant Collection Program. And he will talk to us

    15    today about granulocyte product evaluation.

    16                DR. ADKINS:  Okay.  I'd like to thank

    17    Liana Harvath for inviting me to discuss our data from

    18    St. Louis on granulocyte transfusions that are

    19    mobilized with G-CSF.  Could I have the first slide? 

    20    Liana has asked me to discuss product evaluation in

    21    this area, and I will focus on cell dose and leukocyte

    22    compatibility.  Because at least at this point in time

    23    in my opinion, these are perhaps the two most

    24    important issues in this area today.  

    25                I don't have any specific recommendations

                                                                        155

     1    about either of these two issues, but I would suggest

     2    or put a vote in to provide additional research

     3    support to determine efficacy parameters as they

     4    relate to product evaluation.  

     5                In my opinion, granulocyte transfusions

     6    may be the only strategy which has the ability to

     7    eliminate severe neutropenia after high dose therapy,

     8    which is obviously the reason to pursue this area.  It

     9    is important to ask what are reasonable goals to

    10    achieve with granulocyte transfusions.  Certainly it

    11    would be nice to show that these products reduce

    12    febrile days, antibiotic requirements, reduce the

    13    occurrence of documented infections, and to

    14    successfully treat documented infections as we have

    15    discussed.  Perhaps in high risk populations, one

    16    might be able to demonstrate reduced mortality.

    17                I think as you have already seen this

    18    morning, it can be very difficult to demonstrate these

    19    efficacy parameters in clinical trials.  There are

    20    many causes, for instance, of febrile days beyond just

    21    an infection.  Transfusions can cause that,

    22    antibiotics, et cetera.  So it introduces a lot of

    23    confounding variables in trying to set up a well-

    24    designed clinical trial in this area.

    25                Why use granulocyte transfusions to

                                                                        156

     1    prevent neutropenic infection related complications? 

     2    Well, certainly there are preclinical models, as you

     3    know, that demonstrate a clinical benefit in terms of

     4    improve survival with granulocyte transfusions.  In

     5    addition, there are two meta-analyses of randomized

     6    human trials that also demonstrated a clinical

     7    benefit, but only if certain conditions were met. 

     8    This is data from two decades ago published by Fred

     9    Applebaum demonstrating the critical importance of

    10    component cell dose on efficacy.  In this case, which

    11    is a model of neutropenic dogs with Pseudomonas

    12    bacteremia, this data demonstrated the steep dose

    13    response curve that they observed in this trial.  And

    14    if one was able to infuse products containing more

    15    than 2 times 108 per kilogram, uniform survival was

    16    observed.  And interestingly, the same threshold dose

    17    would correlate with significant increments in ANC. 

    18    This kind of data suggests to me that it is important

    19    to demonstrate significant increments in the ANC to go

    20    on to demonstrate measures of efficacy.  And

    21    interestingly, in a 7 kilogram human, this threshold

    22    cell dose correlates with about 1.4 times 1010

    23    granulocytes.

    24                As I mentioned, two meta-analyses of

    25    randomized trials of granulocyte transfusions in

                                                                        157

     1    humans have demonstrated benefit.  In the analyses of

     2    prophylactic granulocyte transfusions, granulocyte

     3    transfusions that contain an adequate dose of

     4    leukocyte compatible components resulted in decreased

     5    relative risk of infection, mortality, and death from

     6    infection.  In the analysis of therapeutic granulocyte

     7    transfusions, these products resulted in improved

     8    survival if an adequate dose was administered and if

     9    they were given to patients with inherently low risk

    10    or low likelihood of survival.

    11                The combination of these trials suggest

    12    that cell dose is a very important determinant of

    13    efficacy and perhaps leukocyte compatibility as well. 

    14    These are trials of non-G-CSF mobilized granulocyte

    15    transfusions.  

    16                Historically, limitations of granulocyte

    17    transfusions have primarily been issues revolving

    18    around low cell dose and the presence of leukocyte

    19    incompatibility.  Both of these factors are probably

    20    the primary determinants or the primary cause of poor

    21    and nonsustained increments in the ANC with

    22    granulocyte transfusions not mobilized with G-CSF. 

    23    Another important problem in prior trials was the

    24    frequent occurrence of febrile transfusion reactions,

    25    a problem which confounded the analysis of efficacy. 

                                                                        158

     1    It is my hypothesis and perhaps that of others in this

     2    room that significant and sustained post-transfusion

     3    increments in the ANC may be a key pre-condition for

     4    demonstrating reproducible improvements in clinical

     5    outcomes with granulocyte transfusions.  So that

     6    became the focus initially of our research in this

     7    area.

     8                Let's look at ways of improving component

     9    yield or component cell dose.  Most people in the last

    10    few years have been focusing on trying to increase

    11    donor ANC, and certainly as Tom Price has shown, that

    12    is probably the most important determinant of

    13    granulocyte component yield.  And so with growth

    14    factors such as G-CSF, we can clearly increase the

    15    donor ANC pre-collection.  There are apheresis

    16    factors, though, as Susan Leitman has eluded to which

    17    we need to work on to try to improve component yields

    18    with apheresis.  I will talk about our data with

    19    varying the interface offset and showing how that

    20    influences component collection efficiency and yield.

    21                This is a table that I put together which

    22    shows you the component granulocyte dose based on

    23    choice of mobilizing agent.  And as has been discussed

    24    earlier, with granulocytes collected at steady state

    25    and without a red cell sedimenting agent, one can only

                                                                        159

     1    collect about .1 to .3 times 1010 cells.  With

     2    corticosteroids and hetastarch, the average collection

     3    contains about 2.3 times 1010 cells but no better than

     4    3 in general.  With G-CSF, it has been our observation

     5    that one can increase component cell doses by a factor

     6    of several fold, number one, and number two, the

     7    larger the dose of G-CSF that we have given to normal

     8    donors, the greater the component yields, as you can

     9    see here.  We have already seen data from Seattle and

    10    also from this institution showing that there is an

    11    added benefit of giving Decadron along with G-CSF and

    12    resulting component yields.  I am aware of at least

    13    one study that has used GM-CSF to collect

    14    granulocytes, but that was an abstracted report which

    15    did not comment on component cell doses.

    16                As I mentioned, although most people in

    17    the last few years have focused on ways of increasing

    18    donor white count as a strategy to increase component

    19    yields, we have also looked at the effect of altering

    20    apheresis parameters.  One that we have looked at is

    21    the interface offset setting.  As Susan mentioned, the

    22    machine -- using the Baxter device, the machines

    23    default setting is 15.  And to my knowledge, there are

    24    actually quite a few centers that do use that

    25    particular setting, even today.  So we did this study

                                                                        160

     1    where we collected granulocytes using three different

     2    interface offset settings varying from 15 to 25 to 35,

     3    and we determined the effect on granulocyte collection

     4    efficiency and granulocyte yield.  These were all 7

     5    liter pheresis and donors receiving G-CSF at 5 mcg per

     6    kilogram along with hetastarch.

     7                So as you can see, as you increase the

     8    interface offset setting, you improve granulocyte

     9    collection efficiency from 40 to 60 percent.  And this

    10    results in improved yield as you can see here.  So our

    11    practice now is to use an IO setting of 35 instead of

    12    our old practice of 15.  

    13                This is data we recently published

    14    demonstrating again, as others have, that significant

    15    and sustained increments in the recipient ANC occurs

    16    with transfusion of G-CSF mobilized HLA matched

    17    granulocyte components.  Our initial  clinical model

    18    was to collect granulocytes from HLA matched sibling

    19    bone marrow donors who were receiving G-CSF and

    20    transfuse these products as prophylaxis against

    21    infection.  The advantages of this model was that we

    22    chose allogeneic bone marrow transplant patients who

    23    had an expected interval of severe neutropenia during

    24    which most indeed developed complications of that

    25    problem.  There was also an available HLA matched or

                                                                        161

     1    leukocyte compatible granulocyte donor, and that

     2    person would be motivated to undergo frequent

     3    granulocyte collections for their sibling.  In my

     4    opinion, prevention of infection may be an objective

     5    more easily reached with granulocyte transfusions than

     6    successful treatment of established infections.  Many

     7    times these are associated with multi-organ failure or

     8    confounding causes for infection which complicates the

     9    interpretation of efficacy trials.

    10                The limitations of this model is that the

    11    donor has to be ABO compatible and the donor must be

    12    HLA matched.  And obviously the donor would have to

    13    agree to undergo additional time commitment to

    14    participate and perhaps somewhat greater risk over

    15    just bone marrow collection.  

    16                The objectives of this study were to

    17    carefully document the kinetics of the recipient ANC

    18    with each granulocyte transfusion.  The donor

    19    underwent bone marrow collection on day zero,

    20    transplant day zero, and then received G-CSF daily for

    21    five days at 5 mcg per kilogram.  They then underwent

    22    alternating day granulocyte collections on days 1, 3,

    23    and 5.  The recipient underwent marrow infusion on day

    24    zero and then received G-CSF daily from day plus 1

    25    until neutrophil engraftment.  Granulocyte

                                                                        162

     1    transfusions were given fresh on days 1, 3, and 5, and

     2    then we carefully determined ANCs pre and post-

     3    transfusion as you can see here.

     4                This is a table demonstrating for you the

     5    increments that we observed after transfusion of these

     6    granulocyte products on days 1, 3, and 5.  You can see

     7    here that again these are increments.  This is the

     8    mean one hour and mean peak increments that we

     9    observed, and they are quite substantial with the mean

    10    peak increment being up to 11,095 cells per

    11    microliter.  Interestingly, the peak increment

    12    typically occurred about 8 to 12 hours after the

    13    granulocyte transfusion, not at one hour as you might

    14    intuitively expect.

    15                This table demonstrates for you that the

    16    increments were sustained with the time after the

    17    transfusion in which the mean ANC was above baseline

    18    being at least 25 hours.  So we ask the question,

    19    where do these granulocytes go once transfused.  So we

    20    took samples of the granulocyte components collected

    21    on day +5.  We labeled them with indium and then

    22    transfused them into the allogeneic bone marrow

    23    transplant patient and we monitored scintographic

    24    scans serially.  This is a scan obtained within four

    25    hours of granulocyte transfusion demonstrating very

                                                                        163

     1    intense uptake in the lungs.  24 hours later, you can

     2    see that the lung uptake has essentially dissipated,

     3    with intense uptake now in the liver and spleen and

     4    the marrow.  So it looks like these granulocytes

     5    initially, once infused, immediately track for the

     6    most part to the lungs and then are probably gradually

     7    liberated over many hours resulting in that peak ANC

     8    that I mentioned to you earlier.  

     9                We also wanted to know, based on these

    10    kinds of assessments, were these granulocytes

    11    functional, that is, do they localize to sites of

    12    inflammation after transfusion.  Again, this is

    13    another indium scan of G-CSF mobilized and HLA matched

    14    granulocytes.   This is a scan obtained within four

    15    hours of infusion demonstrating predominantly lung

    16    uptake and spleen.  This is a scan obtained 24 hours

    17    later.  Again, the lung uptake has dissipated quite a

    18    bit.  But now you begin to see an area of uptake here

    19    in the cecum and ascending colon.  48 hours later,

    20    this is a very intense area of uptake now, as you can

    21    see outlining the ascending colon and cecum.  This is

    22    a patient who had diarrhea and colitis after their

    23    preparative regime.  We have similar scans

    24    demonstrating this kind of uptake in the mouth in

    25    patients with mucositis.  So these kinds of studies

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     1    along with studies from David Dale and Tom Price's

     2    group would support that these are functional

     3    granulocytes.  

     4                We have just completed a fairly large

     5    study of granulocyte transfusions in the allogeneic

     6    peripheral blood stem cell setting.  The reason we

     7    chose to move from bone marrow to peripheral blood was

     8    really a practical one.  In allogeneic bone marrow

     9    transplantation, the duration of neutropenia is

    10    substantially longer.  And since we are using a single

    11    granulocyte donor, who is the stem cell donor, it is

    12    very difficult to expect them to do 8 collections over

    13    two weeks or three weeks.  So from a practical

    14    perspective, it was better to pursue the allogeneic

    15    peripheral blood stem cell setting where the duration

    16    of neutropenia was about a week shorter.

    17                So in this study, we accrued concurrently

    18    two cohorts, cohort A and cohort B.  Cohort A received

    19    prophylactic granulocyte transfusions on days 3 and 6,

    20    and these were components that were collected from the

    21    stem cell donor who received G-CSF to mobilize these

    22    granulocytes.  Again, they were obviously HLA matched. 

    23    Cohort B did not receive granulocyte transfusions. 

    24    This was a biologic randomization determined by the

    25    availability or not of an ABO compatible, HLA matched

                                                                        165

     1    related donor.  And the donor, as I mentioned, of the

     2    stem cells was also the donor of the granulocytes.  I

     3    have data to share with you on 13 of cohort A. We have

     4    19 actually in cohort A.  And 11 of the 51 we have in

     5    cohort B.  The patients in this study received the

     6    same preparative regimen, the same graft versus host

     7    disease prophylaxis and the same supportive care,

     8    including the way we initiated and stopped

     9    antibiotics.

    10                This demonstrates for you that in cohort

    11    A that received granulocyte transfusions, we did see

    12    significant and sustained increments in the ANC after

    13    the granulocyte transfusions given on days 3 and 6, as

    14    you can see here.  

    15                We then looked at the ANC one day

    16    following granulocyte transfusions in cohort A and on

    17    the same day in cohort B, and we found that the

    18    absolute ANC -- the lowest ANC we observed on that day

    19    was significantly higher in cohort A that received

    20    granulocyte transfusions.  We then tried to determine

    21    an efficacy with this preventive therapy, and we have

    22    looked at numbers of days of IV antibiotics from day

    23    zero until neutrophil engraftment and have observed

    24    that the numbers of days of IV antibiotics in that

    25    interval is about 4.5 days less in the cohort that

                                                                        166

     1    received granulocyte transfusions.  One might argue

     2    that the proportion of patients in cohort B receiving

     3    antibiotics on day zero was greater, and to some

     4    extent that was true because we looked at the numbers

     5    of days of IV antibiotics in the interval from

     6    starting the preparative regimen to day -1, and found

     7    that cohort B had received more antibiotics, but only

     8    by two days, which in my analysis wouldn't account for

     9    this  4.5 day difference we see after day zero.

    10                We do not -- although the absolute numbers

    11    of febrile days was greater in cohort B compared to

    12    cohort A, this was not statistically significantly

    13    different.  That may be more difficult to prove in

    14    this limited number of patients given the relatively

    15    small numbers of febrile days you see here.  So to my

    16    knowledge, this is the first -- this is preliminary

    17    data, but it is the first data that I am aware of that

    18    demonstrates a potential clinical benefit of giving G-

    19    CSF mobilized HLA matched granulocyte transfusions to

    20    such patients.  

    21                With that in mind, we were interested in

    22    knowing the potential importance of leukocyte

    23    compatibility with G-CSF mobilized granulocytes.  I

    24    think one could arguably ask, since we are able to

    25    collect granulocytes with such a huge cell dose today

                                                                        167

     1    using growth factors such as G-CSF, can that massive

     2    cell dose overcome the problem of leukocyte

     3    incompatibility?  So that was the question of this

     4    trial.

     5                This is a study again with the objective

     6    being to determine the kinetics of the recipient ANC

     7    after transfusion of prophylactic granulocyte

     8    components that are mobilized with G-CSF, but these

     9    components may either be leukocyte compatible or

    10    incompatible.  The donor was a first degree relative

    11    of the recipient, received G-CSF daily -- or actually,

    12    I am sorry, four doses on transplant days 1, 3, 5, and

    13    7, and the dose of G-CSF we chose in this trial was 10

    14    mcg per kilogram.  Granulocyte collections were

    15    performed on the evening of day one and the mornings

    16    of day 4, 6, and 8.  The recipients were all

    17    autologous stem cell transplant patients who had

    18    reasonably adequate stem cell products as defined here

    19    based on CD34 numbers.  The recipients received G-CSF

    20    daily from day zero until neutrophil engraftment, and

    21    then received fresh granulocyte transfusions early

    22    morning of day 2 and in the afternoons of day 4, 6,

    23    and 8.  And then we carefully documented the kinetics

    24    of the ANC of the recipient of these granulocyte

    25    transfusions as you can see here.  

                                                                        168

     1                Pre-study, we assessed leukocyte

     2    compatibility between donor and recipient based on HLA

     3    and B typing of both, based on an leukoagglutination

     4    cross match and measures of HLA antibodies using

     5    lymphocytotoxicity assay.  We have accrued 25

     6    donor/recipient pairs in this study, which we have

     7    closed.  I only have data at the moment on six of

     8    these people, which I have shown for you here.  

     9                This is data showing the granulocyte

    10    component cell dose times 1010 for each day of

    11    transplant.  This is the average cell dose. This shows

    12    you the increment in the average ANC at post-infusion

    13    hours 1, 4, 8, 12, 24, 36, and 48.  I think that the

    14    increments that we observed on day +1 were reasonably

    15    good, but the increments that we observed on

    16    transplant days 4, 6, and 8 in my opinion were

    17    inferior to our prior results.  And that occurred in

    18    spite of transfusing larger numbers of granulocytes. 

    19                I show for you here the results of the

    20    leukocyte compatibility test.  Five of six of the

    21    donor/recipient pairs were not HLA and B matched. 

    22    Lymphocytotoxic antibodies were detectable in four of

    23    six patients.  And the leukoagglutination cross match

    24    was zero or negative in all six pairs.  So this

    25    preliminary data suggests to me that leukocyte

                                                                        169

     1    compatibility may be an important determinant of

     2    neutrophil increments after transfusion of G-CSF

     3    mobilized granulocyte products, and it certainly at

     4    least provides an important clue that we really need

     5    to look at this area more carefully.

     6                This is just a table comparing this data,

     7    the current data that I just discussed of autologous

     8    transplant patients who received predominantly

     9    leukocyte incompatible granulocytes. The component

    10    cell dose was 8.6 times 1010, and the maximum mean

    11    peak ANC increment occurring was 796, and that was on

    12    only the day +2 transfusion.  As you recall, the

    13    increments after that were substantially lower.  If

    14    you compare that to our data that we published last

    15    year in Transfusion, these are allogeneic bone marrow

    16    transplant patients who received HLA matched

    17    granulocytes, again mobilized with G-CSF.  In spite of

    18    transfusing a lower component cell dose than this

    19    current data, the ANC increments were substantially

    20    greater.

    21                This brings to me the importance of

    22    considering doing red cell reduction of granulocyte

    23    components.  Why?  When selecting only leukocyte

    24    compatible donors, the requirement of ABO

    25    compatibility  reduces the pool of potential

                                                                        170

     1    granulocyte donors.  And the data from the last study

     2    suggested that we probably need to select leukocyte

     3    compatible donors.  The objective of this study was to

     4    decrease the component of packed red cell volume to

     5    under 5 mls, a guideline established by the AABB,

     6    which we think will reasonably insure against

     7    hemolytic transfusion reaction in recipients of

     8    granulocyte components that are collected from ABO

     9    incompatible donors.  And this is data we recently

    10    published in the Journal of Clinical Apheresis.  

    11                The trial design involved apheresis of

    12    granulocytes with hetastarch, and then after

    13    collection we performed gravity sedimentation of the

    14    component for 60 minutes, and then we transferred the

    15    red cell poor fraction to a sterile docked transfer

    16    bag utilizing a plasma expressor.  The residual red

    17    cells were retained in the collection bag and were

    18    defined as the red cell rich fraction.  And then we

    19    documented cell numbers and packed red cell volumes

    20    with each component or fraction.  This data shows you

    21    that without manipulation of the component, the

    22    average granulocyte component contains a packed red

    23    cell volume of about 25 mls, as Susan Leitman had

    24    discussed.

    25                However, after component manipulation

                                                                        171

     1    using gravity sedimentation ex vivo, the red cell poor

     2    fraction did contain substantially lower packed red

     3    cell volume, 6.3 ml on average.  We observed that in

     4    these manipulated components, 40 percent of the red

     5    cell poor fractions contained under 5 ml packed red

     6    cell volume.  Unfortunately, as I point out here,

     7    there is some cell loss.  20 percent of the

     8    granulocytes are lost to the red cell rich fraction

     9    using this technique.

    10                So ex vivo, a hetastarch sedimentation as

    11    we performed in this study did reduce the number of

    12    red cells from the granulocyte components, but most

    13    red cell poor fractions still contained more than 5 ml

    14    packed red cell volume and thus were not acceptable

    15    for transfusion into ABO incompatible recipients.  We

    16    are currently looking at extending the duration of

    17    sedimentation to 90 and to 120 minutes.  At 120

    18    minutes, we have been able to uniformly reduce the

    19    packed red cell volume to under 5 ml with this

    20    technique.

    21                So I have thrown a slide up here for you

    22    to suggest an ideal trial design for prophylactic

    23    granulocyte transfusion support of transplant

    24    recipients.  The principle being the following:  daily

    25    prophylactic granulocyte transfusions from onset of

                                                                        172

     1    neutropenia to recovery from neutropenia. We would

     2    like to choose leukocyte compatible donors receiving

     3    a large dose of G-CSF.  If you recall an earlier

     4    slide, this dose of G-CSF resulted in granulocyte

     5    components with 20 times 1010 granulocytes.  So there

     6    is a reason for that, which I will get to.  And then

     7    the donors undergo granulocyte collections on

     8    transplant days 2, 4, 6, and 8.  The apheresis uses

     9    the higher IO setting of 35.  Hetastarch is the red

    10    cell sedimenting agent.  And then conceptually, from

    11    a practical perspective, it would be nice to split

    12    each granulocyte component, freshly transfusing half

    13    and trying to store overnight the other half,

    14    realizing that each component will contain roughly 10

    15    times 1010 granulocytes.  It is probably feasible to

    16    do that.  The recipients would include transplant

    17    patients who were receiving adequate stem cell

    18    products, again as I have defined here, and also

    19    receive post-transplant G-CSF.  And it would be nice

    20    to have some measure of choosing non-alloimmunized

    21    patients.

    22                To conclude, first of all G-CSF improves

    23    the cell yield of granulocyte components collected

    24    from normal donors.  Secondly, in allogeneic bone

    25    marrow transplant patients, transfusion of G-CSF

                                                                        173

     1    mobilized HLA matched prophylactic granulocyte

     2    components resulted in significant and sustained

     3    increments in the ANC, localized to sites of

     4    inflammation for up to two days post-transfusion, did

     5    not cause febrile reactions -- and I didn't mention

     6    this, but in our HLA matched granulocyte donor

     7    scenario, we have not observed febrile transfusion

     8    reactions.  These components also resulted in

     9    significant increments in the platelet counts and

    10    reduced platelet transfusion requirements in this

    11    cohort of patients.  And as many of you know, these

    12    granulocyte components contain 2 to 4 times 1011

    13    platelets, which is equivalent to almost a unit of

    14    single donor platelets.  In our experience in this

    15    patient cohort, we have been able to reduce platelet

    16    transfusion requirements in half with granulocyte

    17    transfusions.  Third, antibiotic utilization was

    18    reduced in allogeneic PBSC transplant patients

    19    transfused with G-CSF mobilized HLA matched

    20    prophylactic granulocyte components on transplant days

    21    3 and 6.  Fourth, the preliminary data suggests that

    22    leukocyte compatibility was an important determinant

    23    of ANC increments after transfusion of G-CSF mobilized

    24    granulocyte components.  And fifth, we really don't

    25    know the optimal component cell dose and we really

                                                                        174

     1    don't know the optimal frequency of transfusion of

     2    these products today.  I think we need to learn that.

     3                And finally, there is preliminary data

     4    from Phase II studies suggesting potential clinical

     5    efficacy of G-CSF mobilized HLA matched prophylactic

     6    granulocyte transfusions.  I would suggest that we

     7    give some consideration for pursuing a Phase III trial

     8    at some point to really test this hypothesis.  

     9                I would like to recognize all of these

    10    individuals at Washington University for supporting

    11    these trials.  I would also like to recognize Barnes

    12    Jewish Hospital, which also provided some financial

    13    support for these studies.  Amgen has also been very

    14    kind in helping in doing these studies.  And I would

    15    like to also recognize Gary Spitzer, who provided for

    16    me the initial encouragement to pursue these clinical

    17    trials back in 1992 and 1993.  

    18                Liana asked that I comment on what areas

    19    that I would recommend that we would pursue in terms

    20    of support of research in this area.  I think that we

    21    really need to understand better the importance of

    22    leukocyte compatibility and incompatibility with G-CSF

    23    mobilized granulocyte transfusions.  So I think that

    24    we really need to pursue that issue better and we need

    25    to define that issue better before we pursue a Phase

                                                                        175

     1    III trial.  Secondly, the cell dose is really unclear. 

     2    What is the cell dose we should be using in a Phase

     3    III trial?  I don't think we understand that issue. 

     4    How often should we give these granulocyte components. 

     5    I think that we need to know answers to these

     6    questions before we do a Phase III trial. Red cell

     7    reduction may be an important area to pursue in

     8    research.  Indeed if leukocyte incompatibility

     9    adversely affects outcomes, red cell reduction will be

    10    an important practical issue in order to expand the

    11    available donors that we could choose from.  And

    12    finally, granulocyte storage.  Again, from a practical

    13    perspective, we all would wish that we could store

    14    granulocytes.  It is just, I think, an area that is

    15    probably worthy of considering.  So I will just close

    16    with that point.  Thank you.

    17                DR. STRONCEK:  Thank you for that very

    18    clear and insightful presentation.  He ended with the

    19    right thing to talk about studying next storage.  Tom

    20    Lane will now discuss storage considerations of

    21    granulocytes.  Dr. Lane is Professor of Pathology at

    22    the University of California, San Diego.  He is the

    23    Medical Director of their transfusion service and

    24    their stem cell laboratory.

    25                DR. LANE:  Thank you, David. And thank

                                                                        176

     1    you, Liana, for inviting me to this conference.  I

     2    have learned a lot and it has been very interesting. 

     3    It is always a pleasure to talk about granulocyte

     4    storage.  I will, of course, indicate that many of the

     5    people in this room contributed to the studies that I

     6    am going to summarize.  Give me the first slide,

     7    please.  Were I to actually go through all the data --

     8    the first two slides are actually Dr. Harvath's.  So

     9    if you will move on to the third slide in that

    10    carousel, that starts mine.  

    11                So were I to actually go through all the

    12    data, we would be here all afternoon.  So I am going

    13    to summarize some data that has been collected over

    14    the years regarding granulocyte storage.  I think the

    15    previous speaker has already answered this question

    16    for us, why should we store granulocytes or why should

    17    we know about the storage of granulocytes?  And the

    18    answer to that is for one thing, as Jeff McCullough

    19    has said for years, granulocytes are inevitably stored

    20    for at least some period of time prior to transfusion. 

    21    This relates to variables such as off-site harvesting,

    22    the testing requirements as Dr. Price talked about,

    23    who basically summarized my entire talk by saying that

    24    there are some defects associated with granulocyte

    25    storage, transportation issues, and patient

                                                                        177

     1    considerations.  The patient may be receiving other

     2    forms of therapy that at least some people may

     3    consider incompatible with getting granulocytes at the

     4    same time, such as amphotericin.  Whether or not that

     5    does make a difference.  Or the patient may just not

     6    be able to get the granulocytes when they are ready in

     7    the transfusion service.  And, of course, there are

     8    those of us who actually still do research on

     9    neutrophil function and storage becomes an important

    10    part of that.

    11                But certainly for a clinical trial in

    12    which it may be of interest to store one portion of a

    13    granulocyte preparation, obviously you need to know

    14    whether or not they work.  So I thought I would

    15    summarize this as others have, and I will go through

    16    this quickly.  What the critical granulocyte functions

    17    are.  Obviously, granulocytes need to stay in

    18    circulation for a period of time.  They need to be

    19    capable of a certain amount of adherence but not too

    20    much adherence until they encounter an activated

    21    endothelium.  They need to recognize the activated

    22    endothelium, a chemotactic gradient as indicated here

    23    by these little dots.  They need to then migrate

    24    through a chemotactic gradient towards this happy

    25    bacterium that unbeknownst to him has been opsonized

                                                                        178

     1    by antibody and complement.  They then need to engulf

     2    the bacterium and finally kill it.  So these are

     3    critical granulocyte functions that must be

     4    accomplished.

     5                Well, what do we know about the clinical

     6    efficacy of stored granulocytes.  In fact, there are

     7    no studies out there that will attest to the clinical

     8    efficacy of storage of granulocytes.  If you look at

     9    the seven studies that people refer to, those that

    10    were reviewed by Dr. Strauss, and look in the methods

    11    sections regarding storage,  you will find that the

    12    granulocytes were transfused either immediately or

    13    within 4 hours or it is not specified.  So at this

    14    time, there are no clinical studies that will attest

    15    to the efficacy of stored granulocytes.  So that has

    16    left us then with looking at surrogate markers of the

    17    efficacy of the function of stored granulocytes. 

    18    These can be easily broken into two general

    19    categories, in vivo studies and ex vivo studies.  The

    20    former include granulocyte recovery, kinetics and

    21    survival, and distribution, either measured by

    22    isotopic techniques, migration into the buccal cavity

    23    as Dr. Price mentioned, or a more classical skin

    24    window chamber studies of a variety of different

    25    types.  Or what have been measured more frequently

                                                                        179

     1    because they are simply easier and don't involve human

     2    studies or human manipulation, I should say, are

     3    looking at the cell numbers after storage, adherent

     4    function, chemotaxis function, and the other functions

     5    that I mentioned that are critical for granulocytes to

     6    do what they are supposed to do, which is to kill

     7    invading microorganisms.

     8                Well, if you look at all these potential

     9    surrogate markers, which ones are really relevant to

    10    clinical efficacy?  This is actually a fairly

    11    difficult question to answer.  Let me back up for a

    12    moment and say I think clearly the number of

    13    granulocytes that need to circulate in vivo has been

    14    studied.  There are classical studies by Bodie and co-

    15    workers in the late 1960's suggested that we need in

    16    the range of about 500 per microliter.  Now that may

    17    be a facile measurement of something more complicated

    18    such as the total granulocyte storage pool, but there

    19    is at least evidence to suggest that if the

    20    circulating granulocyte level is above 500 that people

    21    are less susceptible to infection if they are

    22    functioning normally.

    23                So apart from the cell number, what else

    24    do we know about how ex vivo function relates to the

    25    susceptibility for infection? At least -- I don't

                                                                        180

     1    think you can come to any firm conclusions, but you

     2    can get clues from, if you will, experiments of

     3    nature.  The two that I have listed here are leukocyte

     4    adhesion protein deficiency of the beta 2 integrins

     5    and chronic granulomatous disease.  This defect

     6    relates primarily to the adherence of neutrophils,

     7    LAD, and CDG, as you all know, relates to the failure

     8    of granulocytes to generate toxic oxygen radicals. 

     9                Now if you then separate patients with LAD

    10    into severe, moderate and mild and then look at

    11    various functions that are correlated with these

    12    clinical defects, you can come up with at least some

    13    generalities.  And likewise with CDG.  And rather than

    14    going through all this, I have summarized that on the

    15    next slide.  All this data put together suggests that

    16    if skin window migration, primarily generated through

    17    looking at LAD deficient patients, is less than 80

    18    percent of normal, this is associated with at least

    19    mild defects in resistance to infection.  Likewise, in

    20    vitro chemotaxis defects, less than 70 percent of what

    21    passes for normal -- and anyone who has done this

    22    knows that this can be quite variable -- have been

    23    associated with infection.  Adherence less than 50

    24    percent has been associated with infection. 

    25    Phagocytic activity of less than 40 percent, microbial

                                                                        181

     1    killing of less than 25 percent, and oxygen radicals,

     2    surprisingly you need very little in the way of oxygen

     3    radical generation to sustain normal microbial killing

     4    function.

     5                So this gives us a clue then as to what

     6    kinds of surrogate markers are going to be useful to

     7    study when you look at granulocyte storage. Again,

     8    commenting on the fact that while perhaps the best

     9    studies are those related to in vivo function, that is

    10    to say do the cells migrate or localize to sites of

    11    infection, recognizing that those are difficult to do,

    12    most people are going to look at least initially at ex

    13    vivo functions.  And then the next question you want

    14    to ask is well looking at ex vivo functions based on

    15    all of the relevant past experience, how do these

    16    functions fall out?  And nearly all studies agree that

    17    chemotaxis, the migration of neutrophils, is the

    18    single most sensitive function during storage.  It is

    19    the function that seems to have the earliest defects

    20    and is most sensitive to granulocyte manipulations in

    21    storage.  And that seems to be followed by changes in

    22    adherence and microbial killing, followed in turn by

    23    changes in phagocytosis and oxygen radical generation.

    24    So the next studies that I am going to summarize will

    25    focus to a large extent on chemotactic function. 

                                                                        182

     1    Because arguably if the cells won't migrate, then it

     2    doesn't matter if they have 70 percent or 100 percent

     3    oxygen radical activity.

     4                So what are the important factors we need

     5    to look at when looking at stored neutrophils?  What

     6    factors may affect the quality of stored neutrophils. 

     7    Obviously the donors presumably will have normal

     8    neutrophil function and that is usually ascertained by

     9    whether or not they have a history of infection. 

    10    Otherwise, they wouldn't be normal donors.  Obviously

    11    related to the use of G-CSF, donor treatment with G-

    12    CSF or steroids.  Collection techniques as has been

    13    summarized by Dr. Dale.  There are differences in the

    14    efficacy of granulocytes based on whether they are

    15    collected using centrifugal techniques or filtration

    16    leukopheresis techniques.  The concentration of the

    17    neutrophils in the component itself -- and this is

    18    perhaps the single most important thing I am going to

    19    say today.  We are going to have to be careful in the

    20    modern age regarding the concentration of neutrophils

    21    in the bag as regards their storage.  And others have

    22    shown that the concentration of platelets too may

    23    affect the storagability of granulocytes.  

    24                Physical parameters of storage including

    25    temperature, agitation, the type of container, and

                                                                        183

     1    metabolic parameters such as the anticoagulant used,

     2    the pH of the medium, which relates of course to the

     3    concentration of neutrophils, and the amount of

     4    glucose in the protein have been shown to affect

     5    granulocyte storage.  And finally, the presumed

     6    effectiveness or lack thereof of preservatives such as

     7    growth factors.  A letter to the editor regarding the

     8    use of gels to protect granulocyte function.  

     9                I may, in the interest of time, skip this

    10    slide since Dr. Dale has already reviewed the fact

    11    that cells collected by filtration leukopheresis can

    12    be shown up front to have diminished function and this

    13    simply illustrates that while cells collected by

    14    centrifugation leukopheresis at least after collection

    15    are normal -- and by the way, everything that I am

    16    going to talk about will be regarding donors not

    17    stimulated by G-CSF.  So while cells collected by

    18    centrifugation leukopheresis are relatively normal,

    19    those by filtration are not and survive in storage

    20    very poorly.

    21                What about storage conditions and how this

    22    relates to function of stored neutrophils?  Well, very

    23    early studies by McCullough and co-workers have shown

    24    that different kinds of bags may affect storage. PVC

    25    bags appear to be the best of those looked at, at

                                                                        184

     1    least at that time, and are certainly better than

     2    storing neutrophils in tubes.  More recently, TOTM,

     3    and I can't remember right now what this stands for,

     4    or the CLX bags have been shown to provide improved

     5    granulocyte storage compared to other types. 

     6    McCullough and co-workers looked at a variety of

     7    different kinds of anticoagulants and found some

     8    differences which I will show in a moment. 

     9    Sedimenting agents have been largely shown not to

    10    affect granulocyte storage.  I will talk about

    11    temperature, agitation, neutrophil count.  Platelets

    12    I have already mentioned.  Glasser and colleagues

    13    showed some years ago that platelets -- the presence

    14    of platelets diminishes granulocyte function after

    15    storage as a continuous variable, largely through the

    16    diminution, they found, of glucose content.  Likewise,

    17    glucose content of the storage medium somewhere

    18    between 50 and 1,000 mg per deciliter appears to be

    19    optimal.  Glasser and colleagues also showed that

    20    there is a requirement for optimal storage for protein

    21    and found that 1 percent albumin or plasma were

    22    equally effective, but not IgG.  Other studies have

    23    shown a pH optimum in the range of 7 to 7.5 in

    24    preserving chemotactic function.  I am going to just

    25    show you a little bit of that data now.

                                                                        185

     1                This is a study by McCullough and co-

     2    workers published quite a long time ago which showed

     3    for granulocytes stored at 4 degrees Centigrade in

     4    those days a slight advantage at 24 hours to cells

     5    stored in ACD or CPD anticoagulant compared to heparin

     6    or ion exchange.  Not a great deal of advantage but a

     7    little bit.  

     8                For pH, McCullough and co-workers also

     9    looked at this as have others.  Shown here is the

    10    initial pH of a storage medium now 24 hours at room

    11    temperature and the resultant ATP.  This is

    12    chemiluminescence, the measure of toxic oxygen radical

    13    generation and chemotaxis using, I believe, a Boydian

    14    chamber technique.  They found, focusing on

    15    chemotactic function, that a pH range between 7 and

    16    7.5 was optimal.  Either side of the chemiluminescence

    17    dropped off radically.  This illustrates that

    18    chemiluminescence or the ability to generate toxic

    19    oxygen radicals in response to a phagocytic stimulus

    20    was somewhat more resistant to changes in pH and ATP

    21    showed decrements in pH at anything other than about

    22    7.5.

    23                This slide summarizes two studies relating

    24    to the effect of agitation.  It is interesting that

    25    there are differences.  The first one by McCullough

                                                                        186

     1    back in 1978 suggested that granulocytes stored at

     2    room temperature for 24 or 48 hours had a greater

     3    defect in chemotaxis if they were stored agitated, and

     4    Mary Clay was kind enough to remind me that this was

     5    using horizontal agitation, and these changes were

     6    more pronounced after 48 hours of storage. So there is

     7    an advantage to not agitating neutrophils according to

     8    these studies.  But approximately 9 years later, some

     9    Japanese workers published in Transfusion just the

    10    opposite result using a somewhat different bag but the

    11    same kind of agitation, that is to say horizontal. 

    12    They found chemotactic function better preserved in

    13    cells that had been agitated as opposed to left

    14    stationary.  Now it is difficult -- looking at these

    15    two studies, it is difficult to make any sense out of

    16    this since most of the other factors relating to the

    17    granulocyte storage were relatively equal.  What is of

    18    interest is that in the later study, the cells that

    19    were stored in a stationary fashion had a very, very

    20    marked defect in chemotaxis after 24 hours, much

    21    greater than most other workers have found, and this

    22    leads one to believe possibly that there may have been

    23    something else going on in this study which didn't

    24    permit these cells to function as well.  I just point

    25    this out to indicate that maybe this is something we

                                                                        187

     1    need to look at again in view of this controversial

     2    data.

     3                A great deal of work has focused on the

     4    temperature at which we ought to store granulocytes,

     5    and this is a study again by one of the leaders in

     6    this field, McCullough and co-workers, looking at room

     7    temperature versus 6 degree storage for 8 or 24 hours

     8    and looking at in vivo recovery and survival and

     9    localization, this percent skin window migration.  To

    10    summarize this, these investigators found after only

    11    8 hours of storage at either room temperature or 6

    12    degrees, a significant benefit to room temperature

    13    storage in terms of overall granulocyte recovery, and

    14    this is percent recovery, compared with 6 degrees. 

    15    And likewise after 24 hours of storage, again an

    16    advantage to room temperature over 6 degrees storage. 

    17    The half-life measurements of granulocytes were a

    18    little bit more difficult to interpret, but again

    19    suggested an advantage to room temperature storage. 

    20    But these investigators found, again, a marked

    21    advantage to room temperature storage compared to 6

    22    degrees in looking at migration of the cells into skin

    23    windows.

    24                Some studies we did later suggested a

    25    possibility for the changes in granulocyte recovery

                                                                        188

     1    comparing 6 degrees to room temperature storage.  We

     2    looked at granulocyte adherence to endothelial

     3    monolayers after 24 and 48 hour storage of

     4    granulocytes at these two temperatures and found that

     5    cells stored at 6 degrees were somewhat hyperadherent,

     6    whereas those stored at room temperature for this

     7    period of time had relatively normal adherence

     8    function.

     9                We also, as have others, found a

    10    significant benefit to room temperature storage

    11    compared to 6 degrees in terms of chemotactic function

    12    and this is distance migrated shown on the ordinate

    13    scale at 24 and 48 hours, room temperature versus 6

    14    degrees, and this is random migration.  

    15                So this slide then summarizes a number of

    16    studies looking at room temperature versus 4 degrees

    17    Centigrade storage in granulocytes.  Again,

    18    highlighting the differences, there are differences in

    19    adhesive function of cells stored at room temperature

    20    versus 4 to 5 degrees, improvements in chemotactic

    21    function or I should say less of a decrement in

    22    chemotactic function and less of a decrement in the

    23    recovery after transfusion, and less of a decrement in

    24    skin window migration.  So these and other studies led

    25    most people to the conclusion that room temperature

                                                                        189

     1    storage for these unstimulated donors was superior to

     2    6 degrees.  

     3                Someone asked the question today about the

     4    effects of irradiation.  I have summarized here I

     5    think it is 8 different studies that have over the

     6    years looked at the effect of irradiation on

     7    granulocyte function.  And I think we can summarize

     8    these by saying that over the range of irradiation,

     9    these are in gray, used to prevent GVHD, there really

    10    is no consistent effect on granulocyte function,

    11    either in cells that are collected freshly, as most of

    12    these were, or after storage for 24 or 48 hours here

    13    in two studies in which a wide variety of functions

    14    were observed.  There is only one study that suggested

    15    that 50 Gray irradiation might decrease the nitroblue

    16    tetrozolium generation in granulocytes.  And this is

    17    using a semi-quantitative technique.  So I think the

    18    great weight of evidence suggests that irradiation

    19    does not affect these cells.  Again, this hasn't been

    20    looked at in G-CSF stimulated donors, at least not

    21    published to my knowledge.

    22                So using the "best of current techniques",

    23    what can we expect for granulocytes that have been

    24    stored for 24 or 48 hours.  That means room

    25    temperature storage, not agitated.  Well, we can

                                                                        190

     1    expect about 50 percent recovery, which is not very

     2    different from normal -- I am sorry, 50 percent

     3    decrement in recovery compared with normal, which is

     4    decreased, whereas the survival of the cells will be

     5    close to normal.  In vitro, that is to say in the bag,

     6    we can expect to recover most of the cells.  Most of

     7    them will be there, between 99 and 88 percent.  We can

     8    expect up to 20 percent decrement in in vitro

     9    chemotaxis and perhaps 10 percent decrements in

    10    microbial killing.  So all of this data suggests then

    11    that these cells ought to function relatively normally

    12    once they are transfused.  At 48 hours of storage that

    13    may not be the case.

    14                The next and final thing that I want to

    15    talk to, and I am going to try not to go too much over

    16    time here, is I think important to the modern

    17    situation.  Because we did some studies some years ago

    18    looking at the effect of granulocyte concentration,

    19    that is to say the number of granulocytes or their

    20    concentration in the bag, on the subsequent function

    21    of those cells.  We looked at three different cell

    22    concentrations, 2, 5, and 7 times 107 per ml.  We

    23    stored the cells at room temperature for 24 or 48

    24    hours in autologous plasma unagitated at room

    25    temperature.  We found progressive decrements in ATP,

                                                                        191

     1    in glucose, and marked changes in the pH of the

     2    surrounding medium.  We found that -- well, let me

     3    summarize this.  We found basically that you could

     4    prevent the changes in pH by adding 15 millimolar

     5    bicarbonate.  Let's start with this box over here or

     6    this panel.  Shown here are cells stored at -- let's

     7    see, it doesn't say it on here -- at 8 times 107 per

     8    ml, either in the presence of no additives, of glucose

     9    alone, of bicarbonate, or glucose and bicarbonate. 

    10    And you can show that bicarbonate preserves the

    11    starting pH of these cells.  Glucose will preserve the

    12    glucose content, but as shown here will not preserve

    13    the pH.   And that likewise cells stored in the

    14    presence of bicarbonate, either with or without

    15    glucose, maintained their content of ATP, at least at

    16    relatively normal amounts.

    17                Now how does this relate to their

    18    function.  In another study, we looked once again at

    19    ATP content and the pH of cells stored at 2 or 8 times

    20    107 neutrophils per ml, again in the presence of

    21    bicarbonate or without it.  I think if we focus on

    22    this panel, these are cells stored at 8 times 107 per

    23    ml in the absence of bicarbonate.  You can see that

    24    their chemotactic function now using a Boydian chamber

    25    technique drops off rather remarkably.  But in the

                                                                        192

     1    presence of bicarbonate -- unfortunately I don't have

     2    the key here, so I am having to remember these old

     3    studies -- in the presence of bicarbonate, you can

     4    preserve chemotactic function at least for 24 hours,

     5    even at cells stored at this high a concentration. 

     6    Likewise, you can preserve pH and ATP as I showed

     7    before.  So the point of all this is that in the cells

     8    that are being generated and the granulocyte

     9    concentrates that are being generated today, if you

    10    translate the doses that you are giving in the range

    11    of 40 billion or so versus the volumes into which they

    12    are being collected, you are exceeding even what we

    13    studied here by two or three-fold in terms of overall

    14    cell concentrations.  So if we found marked defects in

    15    pH maintenance and ATP maintenance and chemotaxis in

    16    cells stored at 8 times 107, then cells stored at 20

    17    times 107 are probably going to be much worse off than

    18    we have here.  

    19                So I would hasten to add that we really do

    20    need to study how these cells will function after

    21    storage.  And perhaps I would recommend then that if

    22    one is going to try to store these cells, that you

    23    need to try to maintain the pH of the surrounding

    24    medium.  That seemed to be accomplished by using 15

    25    millimolar bicarbonate, at least in these studies. 

                                                                        193

     1    But I would not speculate whether that would be enough

     2    in cells stored at two or three times this

     3    concentration.  I think that is an area that really

     4    needs to be looked at.  And as an abstract to be

     5    presented later on shows, in fact the changes we found

     6    in pH here certainly are found in cells in stimulated

     7    donors.

     8                So finally, I want to summarize here what

     9    everybody already knows.  Over the past several years

    10    there have been a number of studies that suggest that

    11    certain cytokines can, in fact, prolong the storage

    12    life of neutrophils.  Most of these studies were

    13    performed in cells stored at 37 degrees in tissue

    14    culture flasks or plates and looked only at so-called

    15    viability or trypan blue dye exclusion.  So all one

    16    knew was that these cells were surviving longer.  But

    17    more recently, Rex and co-workers have published in

    18    Transfusion some more interesting results.  Again,

    19    these granulocytes were stored in culture dishes at 37

    20    degrees, but they looked at some more relevant

    21    functions, in this case granulocyte chemotaxis to

    22    FMLP, and found that when you store cells in this

    23    fashion for 20 hours, there is a marked decrement in

    24    chemotaxis that is preserved or prevented by the

    25    addition of G-CSF and gamma interferon.  They also

                                                                        194

     1    looked at superoxide anion generation at fresh and

     2    after storage, and again found a decrement with

     3    storage which was again prevented by G-CSF and gamma

     4    interferon.  They looked at bacterial killing and

     5    found perhaps some improvement with G-CSF and gamma

     6    interferon in this decrement.  Now this is percent

     7    surviving Candida.  This is not really a convincing

     8    difference.  

     9                And then they also provided, as have

    10    others, an explanation for why these decrements in

    11    function were prevented by G-CSF.  This is a slightly

    12    different organization here.  We are looking at cells

    13    stored at 20 hours at 37 degrees as a percent of

    14    control, either unirradiated or irradiated with in

    15    this case I guess it is 5 Grays -- maybe that should

    16    be 50.  They found, looking at apoptosis, that cells

    17    stored in this fashion with or without irradiation

    18    underwent marked apoptotic changes, as others have

    19    reported, but that the addition of gamma interferon

    20    and G-CSF with or without irradiation prevented

    21    apoptotic change.  

    22                Now I used to think of granulocytes as

    23    being end stage cells that didn't do much in the way

    24    of protein synthesis and I really didn't understand

    25    these findings for quite some years, but I have been

                                                                        195

     1    educated.  Granulocytes are, in fact, capable of new

     2    protein synthesis and new messenger RNA synthesis, and

     3    I have just summarized some recent studies here

     4    showing that with these various stimuli, you can in

     5    fact get new protein synthesis, as indicated here, and

     6    this is accompanied by gene activation.  And in fact

     7    in I guess it is the August issue of Blood, there is

     8    a new publication indicating that if you look at all

     9    the different species of messenger RNA that are made

    10    by granulocytes, there are over 700 different species

    11    of messenger RNA that are actively generated by

    12    granulocytes.  So these aren't cells that are

    13    incapable of protein synthesis.  And that perhaps

    14    explains how it is that G-CSF and some of these other

    15    stimuli can modulate the apoptosis of neutrophils.  So

    16    I thought I would end here by showing that a variety

    17    of different cells can modulate or delay apoptosis --

    18    interleukans 2415, GM-CSF, G-CSF of course, TNF alpha 

    19    after prolonged incubation, glucocorticoids, et cetera

    20    -- while other functions, stresses, and agents may

    21    accelerate granulocyte apoptosis.  Well, after being

    22    interested in granulocyte storage for 20 years, maybe

    23    this is the holy grail.  Maybe delaying apoptosis in

    24    these cells will permit them to be stored for a longer

    25    time.

                                                                        196

     1                But once again, I guess if I can say

     2    anything important here this afternoon, it would be

     3    that it doesn't matter whether the cells are apoptotic

     4    or not if their pH is 5.5 and they are dead.  So I

     5    think with the modern collection techniques, we need

     6    to be mindful of the concentration of granulocytes in

     7    the medium and do something about that.  And then in

     8    the future, it may be possible to prolong or permit

     9    longer storage using some of these agents.  And in

    10    response to Liana's question, I am going to sound like

    11    a broken record.  I think we need a trial of the

    12    efficacy of granulocytes stimulated by G-CSF in donors

    13    before we can really know whether they are working. 

    14    I would be -- I think we need to study granulocyte

    15    storage, but I would be hesitant to store granulocytes

    16    in such a trial.  I think we have no current knowledge

    17    that stored granulocytes, at least beyond 6 to 8

    18    hours, really work, and I would be concerned at least

    19    in a major trial that it would not be beneficial to

    20    the potential efficacy of such a trial to include

    21    stored granulocytes.  I think that needs to be studied

    22    separately.  Thank you very much.

    23                DR. STRONCEK:  Thanks, Tom.  It sounds

    24    like once granulocytes are proven to be effective, we

    25    will be busy for quite a while figuring out the best

                                                                        197

     1    way to store them.  The next presentation will be by

     2    Conrad Liles, who will talk about in vitro assays

     3    predictive of product function.  Dr. Liles is an

     4    Assistant Professor in the Division of Allergy and

     5    Infectious Disease of the Department of Medicine at

     6    the University of Washington, Seattle.

     7                DR. LILES:  The title of my talk is as

     8    introduced, and I thank the organizers for inviting me

     9    to this workshop.  It is in vitro assays predictive of

    10    leukopheresis granulocyte product function.  It is a

    11    little bit of a difficult task because that is what

    12    people have been talking about the entire day, but I

    13    am going to try to talk about our studies in

    14    evaluating leukocytes or granulocytes that are

    15    mobilized with G-CSF and then also those granulocytes

    16    during storage and storage plus or minus the

    17    readdition of G-CSF ex vivo.

    18                First of all, I wanted to talk about --

    19    you have seen this slide before, but this is why we

    20    chose a regimen of 300 mcg of G-CSF and 8 mg of

    21    dexamethasone to stimulate our donors in our

    22    granulocyte collections.  So we proceeded to use this

    23    regimen because it seemed to give the maximal ANC and

    24    it seemed to give it within 12 hours.  The addition of

    25    dexamethasone significantly increased the maximal ANC

                                                                        198

     1    induced by the dose of G-CSF alone.  The greatest

     2    mobilization was with 600 mcg of G-CSF and 8 mg of

     3    dexamethasone.  This drug regimen at least overall was

     4    relatively well tolerated by the normal volunteers

     5    that we used in this study.  

     6                The protocol involved treatment, as you

     7    have seen earlier, with 600 mcg here and collection by

     8    leukopheresis.  And then the measurements were cell

     9    numbers and morphology, immunophenotype analysis by

    10    flow cytometry, chemiluminescence, bacteriocidal

    11    function, and then blood and tissue kinetics in these

    12    cells immediately after collection.  The collections

    13    were from 5 donors and you can see here that the mean

    14    number of cells collected was 77 times 109 cells. 

    15    That is after a starting neutrophilia of 28,700 in the

    16    donors.  

    17                First of all, we looked at

    18    chemiluminescence.  This just shows you the luminol-

    19    enhanced chemiluminescence activity of the neutrophils

    20    in these collected granulocyte fractions.  We

    21    evaluated baseline, that is, these were -- after

    22    stimulation of these donors, we went ahead and looked

    23    in their venous blood and looked to see what their

    24    chemiluminescence activity was in response to PMA.  We

    25    also looked at the baseline activity right here prior

                                                                        199

     1    to collecting the actual neutrophils and stimulating

     2    the donors, and then we also looked at the

     3    leukopheresis product.  What we found is that after

     4    stimulating with G-CSF and dexamethasone, you actually

     5    saw a priming effect of the stimulation procedure, so

     6    there was greater chemiluminescence activity or

     7    greater oxidative burst potential in response to PMA

     8    after giving G-CSF and dexamethasone.  But then if you

     9    looked at the cells after leukopheresis, there is

    10    actually a slight detriment.  So the leukopheresis

    11    procedure per se actually impairs the subsequent

    12    oxidative burst, but still it was greater than just

    13    cells obtained prior to the G-CSF stimulation.  So our

    14    conclusion was that the product here had good

    15    respiratory burst activity and would have potential

    16    activity in fighting or in having microbicidal

    17    activity if retransfused.

    18                We did look at bacteriocidal activity and

    19    the staphylocidal assay ex vivo.  We found that the

    20    leukopheresis PMNs that were obtained after G-CSF and

    21    dexamethasone stimulation were just as effective as

    22    baseline PMNs in terms of killing the Staph aureus. 

    23    So the bacteriocidal activity appeared to be fine when

    24    immediately collected.

    25                When we looked at immunophenotype analysis

                                                                        200

     1    of these cells versus baseline cells, you can see that

     2    in terms of L-Selectin, there is a slight decrease,

     3    about half or 50 percent decrement, in the L-Selectin

     4    expression on these cells as compared to baseline

     5    PMN's.  CD11B was about doubled.  CD18 was also

     6    doubled.  We didn't really see a large induction of

     7    CD14 at this dose of G-CSF.  And then CD16, which is

     8    FCgamma R3, was actually decreased on the

     9    leukopheresis cells.  CD32 or SCgamma R2 was actually

    10    about the same.  And then we did see an induction of

    11    SCgamma R2 or CD64, about a doubling there.  What we

    12    concluded from this is that the leukopheresis cells

    13    did have a slightly different immunophenotype, but it

    14    was a favorable immunophenotype, and one -- given that

    15    we had expression of the SCgamma receptors -- one in

    16    which we though the cells would be effective in terms

    17    of normal host defense function.  

    18                We then went on to look at the in vivo

    19    kinetics of these cells when retransfused.  David

    20    already mentioned this earlier today.  When we

    21    retransfused the cells, we saw that they had a

    22    prolonged half-life.  What is not shown here is that

    23    these cells not only circulated with the prolonged

    24    half-life, but they could get to inflammatory tissue

    25    sites.  In other words, we did recover these cells

                                                                        201

     1    from the buccal mucosa when they were relabeled and

     2    then retransfused, and they also migrated to skin

     3    windows effectively.  So that these cells not only

     4    migrated, but they could also migrate to potential

     5    inflammatory sites.

     6                So our results overall show that we could

     7    get a good yield of neutrophils from donors stimulated

     8    with one dose of G-CSF and dexamethasone, and then the

     9    leukopheresis performed 12 hours afterwards.  The

    10    respiratory burst activity -- I didn't show you all

    11    the other respiratory burst activity to different

    12    stimuli, but it was more or less normal or at least

    13    there was significant activity to the point that the

    14    cells would have microbicidal activity.  Bacteriocidal

    15    activity, at least against Staph aureus, was normal. 

    16    Immunophenotype showed increased CD11B and CD18 and

    17    also an induction of CD64.  And the kinetics showed an

    18    increased blood half-life, but also the ability of the

    19    cells to migrate to tissue sites.  So it appeared

    20    overall that these cells obtained from individuals

    21    after a single dose of G-CSF and dexamethasone

    22    appeared to be functional and would be viable

    23    candidates in a neutrophil transfusion program.

    24                So just to summarize again, G-CSF plus

    25    dexamethasone allows much improved neutrophil

                                                                        202

     1    collection and the treatment is relatively well

     2    accepted with a few adverse effects.  Metabolic and

     3    bacteriocidal functions are preserved and the cell

     4    half-life is prolonged.  And we have concluded that

     5    transfusion of these cells to neutropenic patients may

     6    be useful.  

     7                In the second part of this talk, I really

     8    want to talk about storage, because that is really the

     9    frontier at this point in terms of improving the

    10    program.  As you have heard, many blood banks would

    11    not have the capability of collecting on weekends or

    12    routinely on weekends.  So the ability to obtain cells

    13    and then store them for 24 to 48 hours would greatly

    14    facilitate any sort of neutrophil transfusion program. 

    15    So we wanted to see whether or not with the knowledge

    16    that we had concerning apoptosis and other storage

    17    variables, whether or not we could come up with a

    18    regimen to show effective storage for 24 to 48 hours

    19    so that this could be adopted if we were to have

    20    neutrophil transfusion programs instituted on a

    21    nationwide basis.

    22                The factors compromising the clinical

    23    utility of granulocyte transfusion therapy have been

    24    discussed, but one of the major factors, as was just

    25    discussed in the talk preceding mine, is that

                                                                        203

     1    neutrophils rapidly undergo apoptosis during storage

     2    in vitro.  And as they undergo apoptosis, functional

     3    activity declines.  Granulocyte products obtained by

     4    leukopheresis are currently transfused into the

     5    recipient as rapidly as possible without storage.  As

     6    soon as that granulocyte product is obtained, it is

     7    usually shipped and transfused as rapidly as possible

     8    without any storage whatsoever.  And this certainly

     9    would hamper any sort of program to be instituted

    10    nationwide.  

    11                If we could get effective storage of

    12    granulocytes, then we could be able to obtain a

    13    leukopheresis preparation and a granulocyte

    14    preparation on a Friday and then use it through the

    15    weekend and then get another donor on Monday, and it

    16    would greatly facilitate the ability to maintain an

    17    absolute neutrophil count in a recipient through the

    18    weekend without undue stress in a blood bank program.

    19                The factors that are known to decrease

    20    neutrophil apoptosis during storage ex vivo include

    21    anaerobic environment.  We actually looked at this and

    22    if you really do store isolated neutrophils, that is

    23    neutrophils that you have obtained by venipuncture and

    24    then culture them or maintain them in RPMI plus 10

    25    percent fetal calf serum ex vivo, the anaerobic

                                                                        204

     1    conditions do prevent apoptosis and do significantly

     2    prolong isolated PMN survival.  However, when we

     3    looked at the leukopheresis product, that is, the

     4    product obtained after G-CSF and dexamethasone,

     5    centrifugation leukopheresis, and then tried to look

     6    at the effect of the anaerobic environment on that

     7    leukopheresis product, we found no significant effect. 

     8    So you can't translate findings that you might see

     9    with isolated PMNs with the actual survival of PMNs in

    10    a granulocyte product.  So anaerobic environment did

    11    not appear to be a viable option to maintain cells

    12    during storage.

    13                We also found that reduced temperature

    14    also reduced apotosis of neutrophils during storage or

    15    during maintenance ex vivo, but if we tried to

    16    translate this at 4 degrees into storage of the

    17    leukopheresis product, we found that this was

    18    impractical.  At 4 degrees, there was a lot of

    19    clumping of leukocytes that could never be

    20    retransfused.  However, when we looked at 10 degrees,

    21    we found no significant clumping of leukopheresis

    22    products at 10 degrees.  So we subsequently tried to

    23    look at whether or not 10 degrees was more effective

    24    than room temperature, and I will get to those

    25    studies.

                                                                        205

     1                It is also known that cytokines,

     2    especially G-CSF and I suppose GM-CSF, are most

     3    effective at decreasing neutrophil apoptosis.  So we

     4    wanted to look to see whether or not the readdition of

     5    G-CSF ex vivo to the leukopheresis product would

     6    further prolong neutrophil viability in addition to

     7    just the viability that we could obtain by reduced

     8    temperature.

     9                Corticosteroids are also known to decrease

    10    neutrophil apoptosis, but we didn't want to add

    11    additional corticosteroids to the product that we

    12    obtained.  And, of course, we couldn't add other

    13    agents like LPS which are also known to decrease

    14    neutrophil apoptosis.  

    15                So we thought the best way to try to study

    16    prolongation of neutrophil survival in the

    17    leukopheresis product -- and this is the leukopheresis

    18    product obtained after G-CSF and dexamethasone -- was

    19    to look at reduced temperature and also the readdition

    20    of G-CSF.  This appeared to be most practical.

    21                So the protocol for our study was to

    22    stimulate donors with 600 mcg G-CSF subcutaneously and

    23    also 8 mg of dexamethasone orally.  Then

    24    centrifugation leukopheresis was performed 12 hours

    25    after stimulation.  Then we looked at storage or

                                                                        206

     1    baseline and at 24 and 48 hours.  We looked at room

     2    temperature as one condition.  Room temperature plus

     3    the readdition of G-CSF with 100 nanograms per ml to

     4    the storage bag.  We looked at 10 degrees and then 10

     5    degrees plus G-CSF added to the storage bag.  

     6                Then we looked at the following

     7    parameters.  We looked at white counts and

     8    differential, respiratory oxidative burst activity,

     9    immunophenotype, staphylocidal activity using a

    10    conventional four plate assay of killing of Staph

    11    aureus, and also fungicidal activity.  In terms of

    12    fungicidal activity, we didn't look at what has

    13    usually been looked at and that is blastochlamydia

    14    killing.  We actually looked at hyphae damage, which

    15    is more relevant for the clinical situation, which I

    16    will get to.

    17                Now in terms of storage of this product,

    18    when you look at the ANC of the product, you can see

    19    that it really doesn't change regardless of what

    20    storage condition that we had.  This is fresh product

    21    here and you can see that there is no significant

    22    difference if the product is stored at 10 degrees or

    23    at room temperature, or if it is stored in the

    24    presence of G-CSF, which is shown not on this slide

    25    but another one right here.  So under any condition at

                                                                        207

     1    reduced temperature, whether it is room temperature or

     2    if it is at 10 degrees, you get preservation of the

     3    ANC in the product, and the addition of G-CSF did not

     4    appear to affect the subsequent storage.

     5                Now next when we looked at the respiratory

     6    burst activity, we used luminol-enhanced

     7    chemiluminescence, which is a rapid and sensitive way

     8    to look at the respiratory burst and allows for a

     9    kinetic analysis.  We used a variety of stimuli.  We

    10    used PMA as a soluble stimulus.  We also looked at

    11    opsonized zymosan as a particle stimulus, and its

    12    activity is primarily related to its ability to bind

    13    and then to be engulfed in the CD11B18.  And then we

    14    also looked at FMLP, and FMLP of course binds to a

    15    cell surface receptor.  I am just going to show you

    16    the PMA and opsonized zymosan results, just because it

    17    gets repetitive if we keep on going through it.

    18                Here if we looked at the storage of this

    19    product, and this is at 24 hours, with the stimulus

    20    being PMA with and without G-CSF at the various

    21    temperatures, you can see that the product is always

    22    better in the baseline activity.  It doesn't appear to

    23    matter significantly, at least at this level, whether

    24    or not the product is stored at room temperature or at

    25    10 degrees.  It also, although there appears to be a

                                                                        208

     1    relative greater benefit here at 10 degrees as opposed

     2    to room temperature, this is probably not clinically

     3    significant because the baseline product is certainly

     4    no better than here and this probably reflects that

     5    initial diminution in the product that I said that we

     6    saw earlier.  That is, after the product is first

     7    collected, it appears to be relatively refractory to

     8    a secondary stimulus, but it regains in response to

     9    that stimulus.  So if we were to look at this, we

    10    would say in terms of overall oxidative capacity, it

    11    is relatively well preserved either at room

    12    temperature or at 10 degrees and the readdition of G

    13    probably didn't make any difference.

    14                Similar effects were also seen at 48

    15    hours.  We won't dwell there.  And it is also seen

    16    with opsonized zymosan as a stimulus.  Here you see

    17    the baseline product and then under any of these

    18    storage conditions at 24 hours, you see enhanced

    19    activity in response to opsonized zymosan.  You still

    20    see this enhancement at 48 hours.  So overall, we can

    21    say under these storage conditions here, oxidative

    22    capacity appears to be preserved and the readdition of

    23    G-CSF may not be necessary to retain that property of

    24    the cells.

    25                Now we also performed immunophenotype

                                                                        209

     1    analysis of the neutrophils during storage.  We looked

     2    at adhesion receptors at CD11B and CD18 and also L-

     3    Selectin.  We also looked at the three IgG CD

     4    receptors, CD16, CD32, and CD64.  We also looked at

     5    CD14.  I would just like to show you the CD16 and

     6    CD32, and CD64 data for simplicity, because I don't

     7    want to have to go through all the other ones here. 

     8    The major point here is that in terms of CD16, CD16

     9    declines as cells undergo apoptosis.  What you can see

    10    here is that although there is a slight decline, CD16

    11    expression is maintained throughout the storage period

    12    or through 48 hours whether or not the cells are

    13    maintained at 10 degrees or at room temperature and

    14    whether or not G-CSF is present or absent.  So what we

    15    would conclude is that storage at reduced temperature

    16    with or without G-CSF maintains CD16 expression,

    17    retains CD32 expression, and also retains CD64

    18    expression.  So that reduced temperature of this

    19    leukopheresed product during storage maintains

    20    cellular viability and a favorable immunophenotype in

    21    terms of FC receptor expression.  A favorable

    22    immunophenotype was also retained if you looked at

    23    CD14 expression or in terms of adhesion receptor

    24    expression.

    25                Now I am not going to show the

                                                                        210

     1    bacteriocidal activity here, because the bacteriocidal

     2    activity was always greater than 95 percent throughout

     3    the study period.  So bacteriocidal activity was

     4    always intact regardless of the storage condition. 

     5    But what is more important when we are considering the

     6    microbicidal activity of a storage product is actually

     7    a fungicidal activity.  Because if we were going to

     8    envision a clinical trial, one would be most concerned

     9    with serious fungal infections in our neutropenic

    10    patients.  That is really where the real problem is in

    11    terms of the oncologic and infectious disease

    12    standpoints.  

    13                I just wanted to emphasize the importance

    14    of opportunistic fungal infections in neutropenic

    15    patients.  Prolonged neutropenia or abnormal

    16    neutrophil function are the major risk factors for

    17    opportunistic fungal infections.  These opportunistic

    18    fungal infections now represent the major cause of

    19    infection-related mortality in bone marrow transplant

    20    or marrow transplant patients.  And of these

    21    opportunistic fungal infections, invasive

    22    Aspergillosis and Candidemia are the most common

    23    opportunistic infections or mycotic infections in

    24    these marrow transplant patients in patients with

    25    prolonged neutropenia.

                                                                        211

     1                Our experience at the Fred Hutchinson

     2    Cancer Research Center in Seattle from 1992 to 1996,

     3    which includes the period -- and this is important for

     4    trial considerations and while you really can't

     5    probably rely on historical controls -- and that is

     6    because our infectious disease prophylactic regimens

     7    have changed significantly and thereby impacted

     8    outcome and survival of patients as compared to years

     9    past.  From 1992 to 1996, this reflects the experience

    10    during fluconazole and ceftazidine prophylaxis.  At

    11    the Fred Hutchinson Cancer Research Center during this

    12    period, only 40 percent of patients who developed

    13    fungemia during neutropenia had clearance of fungemia

    14    within 10 days and survived for four weeks.  More

    15    importantly or just as important, less than 30 percent

    16    of patients during this period who developed invasive

    17    mold infections during neutropenia survived for 12

    18    weeks.  This just emphasizes the importance of these

    19    infections in this patient population.  We can treat

    20    most bacterial infections fairly effectively nowadays. 

    21    The problem of CMVs still exist, but our methods to

    22    control CMV problems are much better than they were 10

    23    years ago.  But fungal infections remain a major

    24    problem and it is a problem that now defies current

    25    antimicrobial prophylaxis and one that is really a

                                                                        212

     1    frontier, I think, of oncology and infectious

     2    diseases.  

     3                Now in terms of the fungicidal activity of

     4    the granulocyte product during storage, we wanted to

     5    look at hyphae or pseudo-hyphae killing.  The reason

     6    being is that hyphae of true molds like Aspergillus or

     7    rhizopus species or pseudohyphae of Candida species

     8    are the predominant tissue forms of opportunistic

     9    fungi during invasive infections.  Usually people look

    10    at Candida or blastocandida, which are easier targets. 

    11    The hard target are the hyphae and pseudohyphae, so we

    12    really wanted to evaluate whether or not the cells

    13    stored under these conditions could actually have

    14    activity against hyphae and pseudohyphae.  To do this,

    15    we employed an XTT assay which measures leukocyte

    16    mediated damage to hyphae or pseudohyphae in vitro.

    17                What we found here -- we will first look

    18    at Candida Albicans.  It is that we looked at this

    19    product that was stored at 10 degrees without the

    20    addition of G-CSF, and we looked at neutrophils

    21    obtained or the buffy coat of patients prior to

    22    stimulation or of donors prior to stimulation, then at

    23    baseline, and then at day 1 and 2 of storage of the

    24    product.  So this is the buffy coat of individuals

    25    prior to getting G-CSF and dexamethasone.  This

                                                                        213

     1    represents the activity of the leukopheresis or the

     2    granulocyte product after leukopheresis on day zero

     3    after individuals received G-CSF and dexamethasone for

     4    stimulation.  And then this is the storage of the

     5    product at 10 degrees without the addition of G-CSF. 

     6    Then we looked at two effector to target ratios, the

     7    effector cell being a leukocyte in the granulocyte

     8    product, and the target being the pseudohyphal form of

     9    Candida Albicans.  And what you can see here is that

    10    the activity against the pseudohyphae is maintained

    11    throughout the storage period.  We see good activity

    12    that is maintained throughout the storage period.  So

    13    these cells even after 48 hours can still mediate

    14    activity against Candida Albicans.  

    15                Similarly, activity is fairly well

    16    maintained against Aspergillus fumigatus hyphae, a

    17    very tough organism or a very tough form of the

    18    organism to kill.  So that we see especially at the 10

    19    to 1 E to T ratio good maintenance of activity

    20    throughout the storage period.  It is certainly

    21    greater at day 1 than day 2, but we still have a

    22    significant level of activity at day 2. 

    23                So what can we conclude from these

    24    studies?  First of all we can conclude that the

    25    granulocyte product obtained by centrifugation

                                                                        214

     1    leukopheresis from donors stimulated with G-CSF and

     2    dexamethasone retain significant functional activity

     3    when stored at reduced temperature for 24 to 48 hours. 

     4    From these studies, we could not conclude whether or

     5    not 10 degrees was better than 22 degrees.  In many of

     6    the assays, 10 degrees looked to be slightly better,

     7    but I don't know if that would be clinically

     8    significant.  So I think that just reduced temperature

     9    -- in other words, storage at room temperature or 10

    10    degrees would be superior to storage at 4 degrees or

    11    at 37 degrees.  Also, the addition of exogenous G-CSF

    12    to this granulocyte product provides at best modest

    13    benefit and probably would not be necessary to

    14    maintain granulocyte products for 24 to 48 hours.  

    15                So what are the important questions I

    16    think for future research in this area?  Well, first

    17    of all, we have done our ex vivo or in vitro analysis. 

    18    Now I think it is important to determine does the

    19    stored granulocyte transfusion product function with

    20    appropriate in vivo kinetics when transfused.  We

    21    should establish this to make sure that products could

    22    be stored if we were going to do a clinical efficacy

    23    trial.  

    24                The most important question, though -- and

    25    this is to prevent the unwise use of granulocyte

                                                                        215

     1    transfusions in the future, and that is actually to

     2    perform an appropriate randomized control multi-center

     3    clinical trial.  We have to know what the appropriate

     4    clinical indications for granulocyte transfusion

     5    therapy are.  We can't really extrapolate from

     6    historical controls because the practice has changed. 

     7    We have better antimicrobial regimens.  So really some

     8    sort of controlled multi-center clinical trial is

     9    necessary to determine what are the appropriate

    10    clinical indications and the specific clinical

    11    indications for granulocyte transfusion therapy.  We

    12    have to know is it going to be effective for invasive

    13    fungal infection or invasive Aspergillosis for mucal

    14    mycosis.  Will it be effective for fungemia or

    15    Candidemia?  Will it be effective for bacteremia in

    16    the neutropenic host?  I actually don't think that we

    17    will see a benefit here just because bacteremia in the

    18    neutropenic host is usually fairly well treated now

    19    with the antibiotics that we now employ in clinical

    20    practice.  Then also another area, as David mentioned

    21    earlier, was neonatal sepsis.  We have to determine

    22    the exact specific indications for neutrophil

    23    transfusion therapy.  If we don't, it will be used

    24    maybe inappropriately in situations and actually could

    25    probably cause much greater harm than good and also be

                                                                        216

     1    an unwise use of resources in the future.  Only

     2    through a randomized controlled clinical trial can we

     3    determine the appropriate indications and avoid that

     4    unwarranted use.

     5                So I would like to acknowledge my other

     6    collaborators during these studies, David Dale and Tom

     7    Price.  Milton Gaviria is a fellow that works with us

     8    and he has been doing a lot of the antifungal assays. 

     9    Then Ellen Roger is a technician who has been working

    10    with David Dale and myself for a long period and she

    11    has been working with the granulocyte storage for the

    12    last several years.  Thank you.

    13                DR. STRONCEK:  I'd like to have the

    14    speakers from this afternoon come up and we can answer

    15    questions.  Dr. Leitman?

    16                DR. LEITMAN:  Thank you.  I have a

    17    question for Dr. Liles.  In a slide shown by you and

    18    earlier by Dr. Dale on the kinetics of in vivo

    19    recovery of autologous labeled G-CSF mobilized

    20    granulocytes, the first column had to do with the

    21    recovery.   And the in vivo recovery, if I read that

    22    right, was lower in the G-CSF treated products, a 65

    23    percent in untreated versus 31 percent, although the

    24    half-life was twice as long, 9.6 versus 20 hours. 

    25    Could you comment on that decreased recovery?

                                                                        217

     1                DR. LILES:  Well, actually -- Tom, would

     2    you want to comment?  Or Tom or David, do you want to

     3    comment on that? 

     4                DR. DALE:  The recovery is lower.  That

     5    is, if you transfuse cells that have been collected by

     6    leukopheresis, you don't get the same percentage of

     7    those cells circulating initially.  I think there is

     8    an element of damage to the cells that occurs with

     9    collection.  And several people have commented that

    10    the one-hour increment in the counts are not

    11    necessarily the highest.  And Tom and I found years

    12    ago that if you collect cells by leukopheresis and

    13    transfuse them, sometimes the counts go up and

    14    actually cells that initially marginated will enter

    15    the circulating pool.  Those are the extrapolated

    16    values you would get at the initial time of

    17    transfusion.  So I think that the values are lower

    18    than normal, but I think the cells probably do recover

    19    some function from being back in the warm, healthy

    20    body.

    21                DR. LEITMAN:  So those are one-hour

    22    recoveries.  But from data presented by all of us here

    23    today, it looks like the 4 or 8 hour post-transfusion

    24    count is higher.

    25                DR. DALE:  That is right.  These are

                                                                        218

     1    extrapolated based on radioisotopic.  In normal

     2    people, not in neutropenic people, you have to use an

     3    extrapolated value based upon isotopic labeling.  But

     4    it is -- would be correct if you could measure the

     5    recovery at four hours later approximately.  I suspect

     6    it would be higher.  Does that make sense?

     7                DR. LEITMAN:  Yes.  I have another

     8    question.  This is for Dr. Adkins.  In your last trial

     9    -- you went through a lot of different trials -- you

    10    are giving allogeneic donors 15 mcg per kilo of G-CSF. 

    11    I want to point out that anytime you exceed 10, that

    12    is two subcutaneous injections because most nursing

    13    standards do not allow you to exceed 1.5 to 2 ml per

    14    single subcutaneous dose, which doubles the discomfort

    15    to the donor to get two subcutaneous shots rather than

    16    one.  The increment in your yield was 15 mcg per kilo

    17    versus 5 mcg per kilo.  It was not very great.  I

    18    think you had 10 times 1010, whereas Seattle and NIH

    19    are getting around 8 times 1010.  And in every study

    20    I have seen, there is a dose-dependent increase in

    21    adverse effects in the donor.  So could you justify

    22    why you are using 15 mcg rather than the lower dose?

    23                DR. ADKINS:  Well, actually in most of the

    24    patients -- the vast majority of the patients in our

    25    clinical trials, of which several you have seen, we

                                                                        219

     1    have used either 5 or 10 mcg per kilogram.  We were

     2    interested in defining whether or not there was a dose

     3    response effect in terms of component yield.  So we

     4    have a very limited number of people that received 15

     5    mcg per kilogram.  In the autologous transplant trial,

     6    which was the latter one that I mentioned, we were

     7    giving 10 mcg per kilogram in that setting.  So I am

     8    not certain that we are going to necessarily pursue

     9    doing 15.  I think you can make an argument.  If you

    10    are going to use a single donor to donate granulocytes

    11    for one patient over a course of a week or so and if

    12    you are thinking of the strategy of storing overnight

    13    a portion of the components, you can make an argument

    14    for using a higher cell dose given that you get

    15    greater yields with that approach.  I mean, that is

    16    just a strategy that one might take and that is

    17    something that we are kind of looking at at Washington

    18    University.  I think that I agree with you about the

    19    issue of toxicity.  You know, we -- in the way we do

    20    this, the donors clearly express a much greater

    21    problem with toxicities during the phase of giving

    22    growth factors to collect stem cells as opposed to the

    23    time in which we give the growth factors to collect

    24    the granulocytes. I don't that the problem with

    25    toxicity is a very big problem when we give the growth

                                                                        220

     1    factors to mobilize the granulocytes.  Their biggest

     2    complaints occur the week prior when we are trying to

     3    collect their stem cells, as I discussed earlier.

     4                DR. SNYDER:  Ed Snyder from Yale.  Just a

     5    couple of practical aspects.  We had done some work

     6    many years ago with stored granulocytes at room

     7    temperature looking at the ability to put them through

     8    an electromechanical pump because many of the oncology

     9    units were doing that to decrease the flow rate and

    10    yet make sure they went in in an appropriate time.  So

    11    studies, if they are going to be repeated with the G-

    12    CSF, that might be a very practical point to look at

    13    to see if the mechanical shear stresses don't have a

    14    negative impact on granulocytes that go through the

    15    pump.  Because some of them can chew up the red cells. 

    16    But we didn't see any problems at that time without G-

    17    CSF several years ago.

    18                Another study that we did was to look at

    19    the effect of the granulocytes on the platelets. 

    20    Because with all due respect to the neutrophils, we

    21    think platelets are also beautiful cells that we need

    22    to care for.  And what we found was that stored for 24

    23    hours at room temperature, there was a decrease -- if

    24    I can remember this and I would have to go back and

    25    look at the paper -- a decrease in GP1B on the

                                                                        221

     1    platelet, which we thought was due to release of

     2    neutrophil enzymes during storage.  So if the FDA is

     3    going to consider neutrophils with platelets in them,

     4    then someone needs to study the effect of storage,

     5    certainly G-CSF stimulated granulocytes on platelet

     6    function or whether the platelets should be removed

     7    from the storage separately if they can co-exist

     8    together in the bag.  So I just mention those for the

     9    record.

    10                DR. DIAZ:   A quick question for Dr.

    11    Liles.  If I interpreted your slide correctly, the

    12    actual kinetics of the response at 48 hours or at some

    13    of the late time points seem to be totally different

    14    from the normal classical response of up and down in

    15    15 minutes and then totalling up to 60 minutes at time

    16    zero.  Can you explain that or did I just read it

    17    wrong?

    18                DR. LILES:  Which one?  The stimulation

    19    with PMA?  You mean the chemiluminescence?

    20                DR. DIAZ:  Yes.

    21                DR. LILES:  I don't have a good

    22    explanation of why that is in terms of why it is

    23    flattened initially.  Is that what you are saying?

    24                DR. DIAZ:  Yes.

    25                DR. LILES:  And then it rises out.  No, I

                                                                        222

     1    don't have a good explanation of why.  It is just that

     2    that was a constant observation.

     3                DR. DIAZ:  Okay, thanks.

     4                AUDIENCE MEMBER:  This is a comment for

     5    Tom Lane.  I liked your presentation in reviewing all

     6    of the details about storage.  I think, though, when

     7    we consider function, we can't go back to and rely on

     8    these congenital defects in neutrophil function to

     9    give us some idea on what we should expect or what we

    10    should shoot for.  An example of this is that you had

    11    stated that we needed to have perhaps 10 percent -- at

    12    least 10 percent or around 10 percent oxidase

    13    activity.  It depends on how you look at it.  If you

    14    look at the kind of classic patients with chronic

    15    granulomatous disease, they have no activity.  If you

    16    look at variants, that data of 10 percent comes from

    17    variants.  And if you have perhaps 10 percent normal

    18    cells, then you will have normal function.  That is

    19    very different than a patient that I have who on a

    20    good day all of her cells have 20 percent activity and

    21    she is always having problems with infection.  And it

    22    should remind us that one of the things that we have

    23    to evaluate in terms of function is whether if we see

    24    decreased function or increased function, whether that

    25    is related to a subset of cells or whether it is all

                                                                        223

     1    cells with lower function or just several different

     2    populations of the function.

     3                DR. LANE:  Yes. I think your point is very

     4    well taken.  That was just meant to give general

     5    information.  The other point that I neglected to make

     6    is that certainly in the presence of multiple defects

     7    in function, even minor defects may take on a lot more

     8    importance. So I think that needs to be kept in mind

     9    as well.

    10                DR. STRONCEK:  Along those lines, though,

    11    I think the issue on storage is very confusing.  I

    12    agree with Tom Lane's summary of the literature that

    13    probably if you are going to do one functional assay

    14    in the laboratory on stored granulocytes, it should be

    15    chemotaxis.  But that said, the literature suggests

    16    that storing granulocytes at less than room

    17    temperature might preserve a lot of function but not

    18    chemotaxis.  I guess my question for you, Dr. Liles,

    19    is have you looked at chemotaxis of your cells stored

    20    at 10 degrees?

    21                DR. LILES:  No.  You know chemotaxis

    22    assays are the most probably variable of all the

    23    assays and that is always a problem.  With newer sort

    24    of techniques, it is possible that you could.  It

    25    might be less laborious.  But I think actually the

                                                                        224

     1    best test is actually to do the in vivo study that we

     2    were talking about and to retransfuse and to see

     3    whether or not you can get proper in vivo migration to

     4    the potential inflammatory site, meaning the skin

     5    window or to the buccal mucosa.  That is really the

     6    real test, even better than in vitro chemotaxis.  So

     7    I think that illustrates the point.  Really we have to

     8    look at these cells when retransfused in vivo to see

     9    whether or not they can adhere to vasculature or to

    10    the endothelium and then migrate through the

    11    endothelium to an inflammatory site.  That will be

    12    most important prior to conducting a large clinical

    13    trial to make sure that stored granulocytes can

    14    migrate to an inflammatory focus.

    15                DR. STRONCEK:  Conrad, a minor point. 

    16    When you talked about bacteriocidal activity, was that

    17    at a one to one ratio? Did you look at different

    18    infectious ratios?

    19                DR. LILES:  Dan, we didn't look at

    20    different ratios.  It was at a one to one at that

    21    point.  So we didn't look under a stress situation, so

    22    to speak.

    23                AUDIENCE MEMBER:  Conrad, I think you

    24    probably mentioned this, but I probably missed it with

    25    all the data.  When you collected these granulocytes

                                                                        225

     1    and stored -- first of all, you collected them on the

     2    CS3000 in what volume?  And then when you stored them,

     3    you just stored them as a product?  You didn't isolate

     4    the granulocytes?

     5                DR. LILES:  Exactly.  So they were the

     6    standard conditions that you saw earlier when Tom

     7    Price discussed it.  So they were stored under those

     8    same conditions and under those same collection

     9    parameters -- yes, COBE.

    10                AUDIENCE MEMBER:  COBE.  All right.  So

    11    what kind of volume do your granulocytes -- what is

    12    the final volume?

    13                DR. LILES:  The final volume is more like

    14    300 cc or so.

    15                DR. LANE:  Again, to ad nauseam.  I am

    16    really concerned about the cell concentrations,

    17    particularly when these cells are stored at room

    18    temperature, and I think that is one of the first

    19    things that people should look at.  And I guess you

    20    are going to present some work on that this afternoon.

    21                AUDIENCE MEMBER:  I would like to reflect

    22    on a comment that was made before and actually second

    23    that.  That is we seem to be coming down to two basic

    24    issues.  One is a clinical trial to look at the

    25    efficacy of granulocytes, and the other big area is

                                                                        226

     1    going to be to look at storage.  And I think probably

     2    we are not going to be able to look at storage -- we

     3    are not going to be able to look at them both at one

     4    time.  It would probably be wiser to not store for a

     5    clinical trial and then come back later when some

     6    basic work is done on storage techniques and look at

     7    that.

     8                DR. LILES:  Could I also make a comment. 

     9    I think also the issue of alloimmunization is very

    10    important.  Because when I am approached by an

    11    oncologist regarding the possibility of granulocyte

    12    transfusions from somebody in the pre-bone marrow

    13    transplant setting, the question is always will giving

    14    granulocytes from community donors obviate the chance

    15    for a successful graft later.  And we really don't

    16    have good information to say whether or not that is

    17    the case.  And that is always going to be a hindrance,

    18    I think, to the use or this practice unless those data

    19    are available. So the issue of alloimmunization and

    20    its clinical importance is still one that is out there

    21    and is worth further investigation.

    22                AUDIENCE MEMBER:  With regards to

    23    alloimmunization and the storage issue, has anybody on

    24    the panel considered cryopreservation, which would

    25    allow you to have autologous donations prior to

                                                                        227

     1    treatment?

     2                DR. LILES:  We tried cryopreservation by

     3    different techniques and you get a gelatinous mess.  

     4                DR. LANE:  I am sorry, I meant to comment

     5    on that and I didn't.  It is very interesting if you

     6    review the literature on cryopreservation of

     7    granulocytes in that you usually find one or at most

     8    two publications by the investigative groups.  Most of

     9    the people who study cryopreservation disappear from

    10    the face of the earth and are never heard of again. 

    11    And that always worried me.  I think there may be

    12    actually a few of us around.  But cryopreservation is

    13    a technology that is so far away from being

    14    practicable for these kinds of cells that it is a

    15    major area that would be wonderful if someone could

    16    find out how to do it, but so far we are very far away

    17    from that.

    18                DR. DALE:  I would just add that what

    19    happens with the best cryopreservation methods now, if

    20    you look by electron microscopy at the cells, they

    21    blub.  And it is probably that the freezing, even in

    22    the best of circumstances, disrupts the membranes of

    23    the granules and you get some damage to the

    24    cytoplasmic components of the cells.  We actually in

    25    the last couple of years have taken this far enough to

                                                                        228

     1    measure chemiluminescence of thawed cells after

     2    freezing, and there is a little activity there.  But

     3    it is really much diminished.  And I don't know where

     4    the breakthrough will come in getting the

     5    cryopreservative into the cell to somehow preserve its

     6    many very fragile parts, but I would not be optimistic

     7    either.  Really the hope is for two or three days of

     8    storage.

     9                AUDIENCE MEMBER:  Well, I think one of the

    10    issues is the clinical situation.  If you look at

    11    something like the red cells where you have the

    12    ability to have wash steps and post-thaw treatments,

    13    you might be in a much better situation than if you

    14    needed something that would be directly transfusable. 

    15    Because there are new cryopreservation programs now

    16    that are looking at water structuring molecules that

    17    cross the membrane that are transfusable and nontoxic

    18    that can, in fact, stabilize organelles and other

    19    components.  So while it hasn't been shown recently

    20    with granulocytes, I think there has been some advance

    21    in cryoprotectant technology that may allow them to be

    22    applicable in this situation.  It certainly appears

    23    that we have a much hardier cell population now also

    24    in terms of these stimulated programs and the ability

    25    to reduce the amount of red cells and platelets in the

                                                                        229

     1    bags.

     2                DR. LANE:  I'd agree by saying that there

     3    is a great opportunity for good work to be done in

     4    this field.  

     5                DR. STRONCEK:  If there are no more

     6    questions, I guess that concludes this part of the

     7    presentation.  We will have abstracts, I guess.

     8                CHAIRPERSON HARVATH:  We have three

     9    abstracts.  Dr. Diaz is here from La Jolla, and Idun

    10    Pharmaceuticals is going to speak on the preservation

    11    of neutrophil viability through inhibition of cast

    12    base dependent apoptosis.  Then after he speaks, Dr.

    13    Stroncek is going to present a couple of abstracts of

    14    their work here that he has done in collaboration with

    15    Dr. Susan Leitman and colleagues at the NIH.

    16                (Whereupon, at 2:59 p.m. off the record

    17    until 3:03 p.m.)

    18                CHAIRPERSON HARVATH:  We will let David

    19    Stroncek present his abstracts first.  They are going

    20    to try and fix the bulb.  Dave?

    21                DR. STRONCEK:  I'd like to thank everybody

    22    who is still here at the end of a long two days of

    23    meetings.  I would like to talk about some studies. 

    24    They are preliminary studies that we have done looking

    25    at first some of the safety aspects of giving G-CSF

                                                                        230

     1    and dexamethasone to people donating granulocytes.  I

     2    have had a lot of experience giving G-CSF to normal

     3    donors, particularly with peripheral blood stem cells. 

     4    And the question comes up during every lecture similar

     5    to the ones we got today about how safe is this really

     6    to be giving donors a drug.  

     7                So with that in mind, I thought it was

     8    worthwhile looking very carefully at granulocyte

     9    donors to determine the effects of G-CSF on them.  We

    10    know from studies on giving G-CSF to stem cell donors,

    11    and we heard some of this this morning, that there is

    12    a marked effect on neutrophils giving G-CSF.  After

    13    the G-CSF is given and stem cells are collected,

    14    platelet counts fall.  The platelet counts fall not

    15    only due to the dropoff from the apheresis collection,

    16    but there seems to be some direct suppression of

    17    platelet production by G-CSF.  In addition, there is

    18    a transient neutropenia and lymphopenia that occurs

    19    after the collection of G-CSF mobilized stem cells. 

    20          We also know in stem cell donors there is a

    21    marked increased in alkaline phosphatase and LDH,

    22    slight increases in uric acid, and falls in potassium

    23    and bilirubin.  These falls are transient but they do

    24    occur predictably in donors.  As you have heard this

    25    morning or today already, both stem cell donors and

                                                                        231

     1    people given G-CSF to donate granulocytes do

     2    experience headaches, bone pain, myalgia, and fatigue.

     3                The question I wanted to focus more on was

     4    what happens with blood counts and blood chemistries

     5    in donors given G-CSF.  In specific, one question that

     6    comes up is because we are using kind of a small group

     7    of people that we have talked to that we have asked to

     8    give G-CSF, we tend to use them over and over again to

     9    donate granulocytes.  One question that I have asked

    10    and we have asked is how often can someone safely

    11    donate granulocytes.  I am not sure what the answer

    12    is, but I do know I would like their blood counts and

    13    chemistries to be normal before we give them G-CSF or

    14    dexamethasone again and collect granulocytes.

    15                So with that in mind, we designed this

    16    study.  We mobilized granulocytes three different

    17    ways.  Either with dexamethasone, G-CSF, or

    18    dexamethasone plus G-CSF.  We used the same protocol 

    19    that Dr. Leitman described earlier today.  The

    20    dexamethasone was an 8 mg dose 12 hours before the

    21    collection.  G-CSF was 5 mcg per kilogram

    22    subcutaneously about 18 hours before the collection. 

    23    And then when we gave both, it was 8 mg of

    24    dexamethasone and 5 mcg of G-CSF per kilogram. Only at

    25    the NIH, but this is a double-blind study.  So, yes,

                                                                        232

     1    we did have placebo tablets or dexamethasone, and we

     2    did either give G-CSF or a placebo injection.

     3                So we did look at symptoms and this was a

     4    nice way to try and sort out the effects of

     5    dexamethasone from G-CSF.  We plan to enroll 24

     6    donors.  I have enrolled 10 so far and I am going to

     7    show you the data on 6.  Each donor would be

     8    randomized to one arm and then we would study their

     9    blood chemistries for several weeks and then they

    10    would come back six weeks later to be enrolled a

    11    second time and a third time.  So all three donors got

    12    each -- all of the donors got each of the three

    13    mobilization regimens and had granulocytes collected. 

    14                What we measured was symptoms, blood

    15    counts, and blood chemistries.  I am going to focus

    16    mostly on the blood count and chemistry data.  We

    17    analyzed the donors prior to mobilization, pre and

    18    post-collection, and then one and two days after the

    19    collection, and then once per week weekly for five

    20    weeks.

    21                This is a summary of the platelet counts

    22    in the donors.  First of all, as you would expect in

    23    all three donor groups, the platelet counts fell after

    24    the collection.  These counts are premobilization. 

    25    And as expected, the counts fell about 20 to 30

                                                                        233

     1    percent.  What we have seen in peripheral blood stem

     2    cell donors is that the platelet counts remain low or

     3    at about post-collection levels for almost a week.  We

     4    didn't see that in any of these groups.  In the

     5    dexamethasone donors, the counts started coming up by

     6    day 4.  And then about a week after collection, the

     7    counts were back into the normal range.  We did see a

     8    slight overshoot in the counts after two weeks.  And

     9    by three and four weeks, the counts were back in the

    10    normal range.  The counts were almost the same in the

    11    people who got G-CSF.  Again, the counts started to

    12    come up -- they were low one day after the collection

    13    and started to come up two days and then a week later

    14    they were back to normal and two weeks after that they

    15    were above normal.  A similar effect occurred with G-

    16    CSF.  So we didn't see any adverse effect of G-CSF on

    17    platelet counts.  It looks like the recovery of counts

    18    is almost identical to donors given dexamethasone.

    19                We also looked at neutrophil counts just

    20    to see if there would be any post-collection

    21    neutropenia.  First of all, as you would expect, the

    22    day 1 neutrophil count is about the same.  They are

    23    the same group of donors.  And as you have seen

    24    several times today, the counts are increased to about

    25    20,000 or more with G-CSF and considerably more in

                                                                        234

     1    people that got G-CSF plus dexamethasone.  The day

     2    after the collection, the counts were still slightly

     3    elevated in the G group and G plus dex, and even

     4    slightly higher at day 2.  We did not -- the next week

     5    and the following week, we really didn't see any

     6    difference in granulocyte counts from the pre-G-CSF

     7    counts.  So based on this data, their granulocyte

     8    counts come back to normal baseline quickly, and at

     9    least a week afterwards, there would be no reason why

    10    a donor could not get another dose of G and donate

    11    granulocytes again.

    12                Concerning blood chemistries, it has been

    13    well known that when you give G-CSF for three, four,

    14    or five days, alkaline phosphatase double or triple. 

    15    A single dose of G-CSF though does not seem to have a

    16    very marked effect on LDH levels.  It does increase

    17    them though. Baseline levels were at 148, 141, and

    18    146, similar in the three groups.  And as you would

    19    expect, dexamethasone did not affect the LDH levels

    20    after the second day.  G-CSF though in both groups did

    21    result in a slight increase in LDH levels.  And then

    22    the day afterward, actually the levels were below

    23    baseline.  I am not sure why that was. It may have

    24    been due to some dilution following the apheresis.

    25                In contrast to peripheral blood stem cell

                                                                        235

     1    donors, we did not see any change in alkaline

     2    phosphatase.  Apparently it takes more than one day of

     3    G-CSF to cause changes in alkaline phosphatase.  

     4                These data were a little bit surprising in

     5    that I did expect the potassium levels to fall when G-

     6    CSF was given, and a single dose did result in quite

     7    a significant drop in potassium from 4.2 to 3.7, which

     8    was back to 4.0 again the day after apheresis.  I was

     9    surprised though to know that a single dose of

    10    dexamethasone can also result in a fallen potassium

    11    level.  The level went from 4.3 to 3.9.  The combined

    12    effect of G-CSF and dexamethasone though does not seem

    13    to be any different than either drug alone.  This

    14    change was very transient and by day 3 the potassium

    15    levels seemed to be almost back to normal.

    16                Another effect I didn't expect with

    17    dexamethasone was a change in albumin levels. With

    18    apheresis, donors tend to get some fluids, and I

    19    thought it wouldn't be unexpected if they would get a

    20    little bit of hemodilution and a fallen albumin.  We

    21    saw that, but we only saw it in donors that got

    22    dexamethasone as opposed to donors that got G-CSF.  So

    23    at least by one measure dexamethasone may actually

    24    cause more change in blood chemistry and G-CSF

    25    doesn't.  That wasn't the only parameter that changed

                                                                        236

     1    slightly with dexamethasone and not G-CSF.  Again,

     2    albumin went up very slightly. Although these numbers

     3    might be statistically significant, I don't think they

     4    are going to make any difference for donors. 

     5    Phosphorus did drop for all three donor groups, but

     6    again transiently.

     7                This kind of summarizes what happens with

     8    the chemistries we measured.  Sodium bicarb,

     9    creatinine, calcium, magnesium, bilirubin, alkaline

    10    phosphatase and SGPT, SGOT, and GGTs were unchanged. 

    11    As I pointed out, all donors in all three groups had

    12    decreases in phosphorus and potassium.  Donors in all

    13    groups had a very slight change in cholesterol and

    14    triglycerides.  Triglycerides might be related to

    15    fasting before apheresis.  I am not sure on the

    16    mechanism of change in cholesterol in the groups. 

    17    Dexamethasone, as I showed, had a slight decrease in

    18    albumin.  And then again we saw the data on the

    19    bilirubin and the LDH.  Chloride was slightly

    20    increased in the dexamethasone group and uric acid had

    21    a very slight increase in all three groups.  The

    22    bottom line is I don't think any of these changes are

    23    really very significant, and I think that based on

    24    these findings it is going to be safe from a blood

    25    count and chemistry point of view to give G-CSF once

                                                                        237

     1    weekly.  

     2                So actually I was quite surprised.  This

     3    data suggests that in some ways dexamethasone might

     4    actually be more potentially harmful to donors than G-

     5    CSF.  So in summary of this part of the data, the

     6    mobilization of granulocytes with either G-CSF or

     7    dexamethasone is associated with mild changes in blood

     8    chemistries.  These blood chemistry changes and cell

     9    counts did return to baseline promptly and it may be

    10    safe for donors to donate mobilized granulocytes at

    11    one-week intervals.

    12                I would like to continue on with the rest

    13    of what I did with this study. On this same study, we

    14    did collect granulocyte concentrates and we did want

    15    to see how well these concentrates stored.  The issues

    16    I think a lot of people have already mentioned today,

    17    though, that granulocyte standards limit storage to 24

    18    hours.  And as we have heard many times, G-CSF

    19    inhibits apoptosis.  It may be possible to store these

    20    mobilized granulocytes more than 24 hours and still

    21    maintain viability and function.  We did have a

    22    concern that high cell concentrations in the products

    23    that we were collecting could counter the effects of

    24    G-CSF and actually diminish viability.

    25                Again, the study was exactly the same. 

                                                                        238

     1    the same mobilization.  We collected the products with

     2    a CS3000 blood cell separator and 7 liters of blood

     3    were processed.  With the CS3000, our volumes tend to

     4    be a little bit less than with the products collected

     5    in Seattle with the COBE.  I will show you later that

     6    the products had about 225 ml of plasma.  We stored

     7    the products at 48 hours, and for this study we

     8    measured cell counts, pH, and we also looked at

     9    viability.  My lab isn't geared up to measure -- not

    10    yet anyway to measure a large number of granulocyte

    11    functions, but as long as we had these products, we

    12    thought we would get some data.

    13                This slide summarizes the first 18

    14    products we collected.  Again, it is very similar to

    15    what Dr. Leitman has shown earlier today.  The volume

    16    of these products are all about the same, 230 ml.  The

    17    white cell concentration though is extremely high in

    18    the products.  For the dex products, it was 144 times

    19    109 cells per liter.  It went up to 204 for the G-CSF

    20    and 332 for the G plus dex products.  I think Tom Lane

    21    showed some data where his highest concentration was

    22    80 times 109 cells per liter.  So this is at least

    23    double that concentration and this is four times that

    24    concentration.  For a lymphocyte culture, we would

    25    usually go with 1 or 2 times 109 cells per liter.  The

                                                                        239

     1    composition of the products, again, was similar to

     2    reported before.  It was 66, 76, and 84 percent

     3    granulocytes.  And these were the total numbers of

     4    cells present, 2.4 times 1010 cells and 3.7 times 1010

     5    and 6.5.  So these are the dose of cells that you have

     6    heard many times that people are transfusing

     7    practically these days.  

     8                The first thing we looked at again was

     9    actual counts on the products, either immediately

    10    after collection, day 1 or day 2.  And as you have

    11    seen before, the counts stayed very steady from the

    12    first day of storage, at least the dexamethasone

    13    products did.  We saw a very slight fall-off in

    14    counts, less than 10 percent after the second day. 

    15    Again, the counts were well maintained in both the G

    16    and the G plus dex products.  So at least by a gross

    17    measure by counts, there wasn't much change in the

    18    number of cells present.

    19                We looked at cell viability using a 7AAD

    20    stain.  The cells were almost 99 percent viable the

    21    first day, maybe 98 percent viable the second day.  We

    22    did -- maybe the fall-off went to 90 to 95 percent the

    23    second day, but really it really didn't change much. 

    24    What we were surprised about though was the pH of

    25    these products.  As Dr. Lane showed earlier, the pH

                                                                        240

     1    was 7.1 to start with.  I think in his most

     2    concentrated products, he had a similar change in pH

     3    at 6.3 the first day and then we went to two days and

     4    it was 6.1.  That wasn't probably too detrimental. But

     5    when you go over to the other products, the very

     6    concentrated products collected from donors given dex

     7    plus G, right immediately after collection the cells

     8    were slightly acidic and a day later the pH was 5.5

     9    and stayed that way the second day.  

    10                The G stimulated products weren't much

    11    better.  They were 6.2 pH after day one and 5.8.  What

    12    was remarkable about these is we saw one or two of

    13    these six products have a little bit of hemolysis of

    14    red cells by the first day and almost all of them had

    15    some hemolysis of red cells present after two days. So

    16    even though these cells might look viable, just based

    17    on the pH and the presence of hemolysis, I wouldn't

    18    transfuse cells with the hemolysis present.

    19                So we think something needs to be done to

    20    try and maintain the viability a little better of

    21    these cells if we are going to store them.  The first

    22    thing we thought of is well maybe it has to do with

    23    concentration.  How far out can we dilute these

    24    granulocyte concentrates in order to maintain the pH. 

    25    This is a summary of preliminary results with diluting

                                                                        241

     1    four concentrates out with autologous plasma.  What we

     2    did was we allocated the concentrates into teflon bags

     3    and added autologous plasma and did a 1 to 2, 1 to 4,

     4    1 to 8, and 1 to 16 dilution.  I had one product that

     5    was -- I lumped the data.  One product was collected

     6    after dex mobilization and one after G and two after

     7    G plus dex.  And what we found was that if we diluted

     8    the product out 1 to 8 or 1 to 16, we began to have a

     9    normal pH and those pH's were maintained in that

    10    normal range even after two days.  So it looks like

    11    diluting a product out just two or four-fold won't be

    12    adequate, but somewhere around 8 to 16 fold might

    13    maintain the pH.  Again, these products were 230 ml to

    14    begin with and we can't collect 2 liters of autologous

    15    plasma.  We are going to try some various additives to

    16    see if that will help maintain storage.  It may be --

    17    we heard other ideas today.  Maybe adding bicarbonate

    18    might be another way to go without diluting out the

    19    concentrate.  

    20                I do agree these are preliminary studies

    21    and we do plan to start to measure chemotactic

    22    activity on some of these stored cells to see if there

    23    is fall-off in chemotaxis function.

    24                So concluding this abstract, to optimize

    25    granulocyte storage, we believe they should be diluted

                                                                        242

     1    6 to 18 fold, especially G and dexamethasone mobilized

     2    concentrates, or at least some other additive should

     3    be added to help maintain the pH.  We will probably

     4    need clinical grade diluents or additives to maintain

     5    the pH. I would like to thank the people that helped

     6    me with these studies.  Dr. Leitman and members of her

     7    apheresis unit, Yu Ying You, Janice Carr, Hatian

     8    Chung, who started some of the assays in the

     9    laboratory with me, and Dr. Tom Lightfoot, who will be

    10    continuing some of these storage studies.  Thank you.

    11                CHAIRPERSON HARVATH:  Are there any

    12    questions for Dr. Stroncek before we start?  Maybe

    13    what we will do is we will hold the question until the

    14    next abstract and we will do it up here.  Sorry,

    15    because that microphone apparently is not working

    16    right now.  Now we will try the overhead projector

    17    once again.  I think we have a new projector.

    18                DR. DIAZ:  So this is the last talk of the

    19    day.  It is Friday.  So I promise there are only 14

    20    slides.   You only have to be awake for 7 of them. So

    21    I think we can get through this very quickly.

    22                Diaz Law Number 2 of presentations.  When

    23    someone from a company is presenting something, its

    24    credibility is inversely proportional to how slick the

    25    presentation is.  So in order to aid my credibility,

                                                                        243

     1    you will see that I have got low-tech black and white,

     2    and in the very first slide I have introduced a

     3    spelling mistake.  Any other spelling mistakes you

     4    spot from now on will be due to my aberrant education

     5    on a small island just off the coast of Europe.

     6                At Idon, our expertise and our interest is

     7    actually in apoptosis.  So the neutrophil is really

     8    just a by-product of what we have been looking at. 

     9    And one of the things that we are very interested in

    10    is in the caspase dependent apoptosis and cell death,

    11    caspases being the enzymes that are involved with the

    12    end stage of the death of cells.  

    13                Very quickly, the neutrophils obviously

    14    have short circulating half-lives in the body.  They

    15    also seem to lose function and dye when stored in ex

    16    vivo, such as in the leukopheresis pack.  We know that

    17    death in circulating neutrophils is apoptotic.  We can

    18    only surmise that perhaps the death that we are seeing

    19    in the leukopheresis packs is also an apoptotic cell

    20    death.  Therefore, what we have been trying to see is

    21    if we can interfere with the normal progress of

    22    neutrophil death by using inhibitors of apoptosis.

    23                So the two inhibitors that I want to look

    24    at very quickly is one which is a generic inhibitor of

    25    caspase, I think it is mostly the IL1 beta converting

                                                                        244

     1    enzyme, which is Z VAD fmk that has been used a lot in

     2    different apoptotic research.  This is one of our own

     3    molecules made by one of our chemists, a highly

     4    experienced and wonderful chemist.  He is my boss, by

     5    the way.  Which is the azile dipeptide fmk, indole

     6    fmk.

     7                So the assays we have been looking at we

     8    set up in order to look at the neutrophils and where

     9    we are really inhibiting some neutrophil apoptosis is

    10    we looked at oxidative burst assays using zymosan. 

    11    The reason why we use zymosan is because probably it

    12    is more physiologically relevant than something like

    13    PMA.  It has been shown that cells that are heavily

    14    into apoptosis can still have some sort of burst with

    15    PMA.  And we have been looking at viability assays

    16    through flow cytometries like hypodiploidy assay,

    17    which is an assay that looks at how intact the

    18    neutrophil nuclei are.  And the other one is annexin

    19    V labeling.

    20                Basically the phosphotidylserine flips out

    21    from the inside part of the cell to the outside part

    22    of the bilipid layer when the cell goes into apoptosis

    23    before it disintegrates.  The other thing we have been

    24    looking at is CD16.  CD16, as Conrad Liles mentioned

    25    before, is the FC gamma 3 receptor.  It has been shown

                                                                        245

     1    in several studies that disappearance of the CD16 or

     2    expression of CD16 in neutrophils seems to correlate

     3    with onset of apoptosis.  

     4                So the series of slides I am going to show

     5    you are all from isolated human neutrophils from

     6    normal donors.  So this is not from apheresis packs. 

     7    This is actually from isolated neutrophils.  And what

     8    we see is that if we look at the time course of the

     9    onset of apoptosis, you can see here that annexin V

    10    without treatment, we actually see very quick

    11    expression of annexin V labeling with

    12    phosphotidylserine on the outside after 24 hours.  By

    13    48 hours, just about every cell has gone.  When we

    14    treat the cell with 1965, this is our sort of

    15    shorthand for that azile indole dipeptide, you can see

    16    that right after 96 hours we have preservation.

    17                We see similar preservation, although it

    18    is not quite as spectacular, when you look at CD16

    19    expression.  So again, by 24 hours you have a huge

    20    diminution of the CD16 being expressed in the cell

    21    surface of isolated neutrophils.  But when you have --

    22    when you incubate these cells in the presence of 1965,

    23    you actually see that there is preservation right out

    24    to 96 hours where still over 60 percent of the cells

    25    are expressing.  Just to show you that these numbers

                                                                        246

     1    aren't made up, we have some pretty colored slides

     2    here.  You can see here that this is the untreated

     3    control at time zero.  You can see here that there is

     4    a nice population in the facts analysis of a

     5    granulocyte population.  It is expressing a nice

     6    homogenous CD16 population, and it is not labeling

     7    with annexin V.  With time, 24, 48, 72, and 96, you

     8    can see there is a very quick drop off of CD16 and

     9    very quick expression of annexin V labeling on the

    10    outside, and you can see that here as well in the

    11    double staining experiments.  Can you see it at the

    12    back?  With the 1965, you see that you do get

    13    preservation.  It is not just a figment of my graphs. 

    14    You can actually see that there is quite good

    15    preservation here.  Although of course you are

    16    starting to see the break-up of the CD16 signal here. 

    17                Of course these are all surrogate

    18    endpoints and we wanted to look also as well at

    19    surface markers to see what do the nuclei of these

    20    cells look like.  So we did the hypodiploidy assay.

    21    This is basically an assay where you, after certain

    22    time points, you allow PI or propidium iodine to enter

    23    into the cell and label the DNA of the cell and then

    24    what you are doing is you do flow cytometry to look at

    25    the cells or the nuclei is still attached as opposed

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     1    to a nuclei that is chopped up.  You can see that out

     2    to this time point, the 1965 protects the integrity of

     3    the nuclei.  The fmk has some slide protection, but it

     4    is not that great, and you can see how this falls off

     5    when it is untreated.  

     6                Perhaps more important is what is

     7    happening to the actual functional part of the cell. 

     8    So we -- this is the oxidative burst, what happens in

     9    the oxidative burst as I say in response to opsonized

    10    zymosan.  I think it is probably the most relevant

    11    since in order for that to form a response, you have

    12    got to have receptors being expressed -- SC receptors

    13    being expressed on the outside to capture the cell and

    14    to capture the opsonized zymosan.  The cell has to

    15    have the ability to restructure its cytoskeletan and

    16    take in phagocytose and then it has to have a

    17    response.  So I think it is a very good way of looking

    18    at the viability of a cell.  You can see that the

    19    ability to burst, isolated neutrophils fall very, very

    20    rapidly after 24 hours and Z-VAD-fmk has very little

    21    protective activity.  The 1965 has protective activity

    22    out to 48 hours, which is actually quite good.  By the

    23    way, I am just showing you -- we have a lot more data

    24    than this, but obviously we just wanted to show you

    25    representative samples.  This is actually at 50

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     1    micromolar.

     2                The IC50 of 1965 for 48 hours is about 10

     3    micromolars. So at 10 micromolar, you are still

     4    getting 50 percent protection.  There are other

     5    compounds that seem to be working even better.  The

     6    1965 is obviously one that we can show you the

     7    structure of.  

     8                So not knowing very much about

     9    leukopheresis or really about neutrophils, we decided

    10    to do something really naive and just put some of

    11    these compounds into an apheresis bag thinking we are

    12    on to a winner here.  If it works on the isolated

    13    neutrophils the way that we are mistreating them, it

    14    is bound to work in the bag.  We set up a series of

    15    assays and in particular set up a series of flow

    16    cytometry assays looking at these different markers,

    17    just so that we could make sure that what we are

    18    looking at are neutrophils.  So in each case we are

    19    labeling with CD3 to identify lymphocytes and CD14 to

    20    identify monocytes, although CD14 is expressed in

    21    neutrophils, it is actually the percentage of

    22    expression is a lot, lot lower than on monocytes. 

    23    CD66B is the expression of neutrophils.  That is a

    24    marker specific for neutrophils.  And then obviously

    25    this looking for platelets.

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     1                In terms of functional markers of cells,

     2    we looked at CD16 and CD16B.  Really, they are both

     3    the same marker.  The only difference is that CD16B is

     4    a isoform of the CD16.  It is actually specific for

     5    neutrophils.  So that is what mainly we are going to

     6    be talking about, CD32.  And then most importantly

     7    also is CD62L, that is L-Selectin.  What we have seen

     8    is that when a cell is activated, it loses its L-

     9    Selectin.

    10                So we did a whole series of experiments. 

    11    Rather than go through all that data, we hit several

    12    problems of inconsistency, which is sometimes we could

    13    get some of our compounds to work and sometimes not. 

    14    We hit a whole series of problems.  And I think a lot

    15    of it were also as we went along what we found was

    16    that we had other issues other than just stopping

    17    apoptosis, which is a lot of these cells were actually

    18    not dying of apoptosis I think in the first place. 

    19    But problems that obviously you are all aware of, but

    20    we weren't aware until very recently, which is

    21    problems of actual mechanical storage of the samples. 

    22    Conrad Liles and other people have obviously gone into

    23    this.  These are things that we started to sort out on

    24    our own.

    25                But really the problem was that we

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     1    couldn't get through the very first hurdle, which was

     2    this.  We were actually collaborating with our local

     3    blood bank, who I don't think really have -- I am not

     4    sure how much experience they have on granulocyte

     5    apheresis products.  But this is the first hurdle we

     6    hit and why we can't give you a straight answer at the

     7    moment as to whether these compounds work.  This is at

     8    time zero.  We actually went through -- I think these

     9    end up being 11 different leukopheresis packs tested

    10    on these days with these different treatments. 

    11    Unfortunately, our local blood bank also didn't have

    12    a G-CSF protocol that we could use.  

    13                So these are the different treatments and

    14    these are the different dates and here is the problem. 

    15    If you look right off you can see there is a huge

    16    variation from week to week on the cell count that

    17    they are actually able to give us.  And then even more

    18    so the neutrophil count.  So the total cell count and

    19    the percentage of neutrophils collected in each time

    20    point is totally different.  

    21                This was a short working day.  I am not

    22    quite sure how that happened but on the 11th of

    23    February we actually got a sample which had no

    24    neutrophils in it whatsoever.  The other problem --

    25    even on the days where we actually had cells and even

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     1    better the days we had cells in the neutrophils, we

     2    found that looking at CD62L there is a huge variation

     3    in the percentage of cells that are actually labeling

     4    with CD62L, which means the state of activation of

     5    those cells is totally different.  We haven't had a

     6    chance to look into this, but I am sure that the state

     7    of activation of the cells varies or influences

     8    dramatically how the cell will survive and whether the

     9    cell is going to go into apoptosis or not.  

    10                Lastly, the viability of the cells --

    11    well, at that particular time, there is not that much

    12    difference.  But I am not quite sure how important

    13    that is or how much it is going to vary.  Really it is

    14    the fact that the number of cells we are collecting

    15    and the site of activation of the cells varied so much

    16    that we can't really go any further with that

    17    particular collaboration. 

    18                So we were hoping to come here so that we

    19    could get answers like how consistent and we have been

    20    pleased with some of the responses here which is how

    21    consistent can we expect the neutrophil apheresis

    22    product to be.  It is obvious that it can be a lot

    23    more consistent.  And then also what state can we

    24    actually expect them to be by the time we get them.

    25                I think the other thing that will be very

                                                                        252

     1    important to us is I think it is going to be very

     2    difficult for us to do anything with apoptosis

     3    inhibitors until we have a system that people agree on

     4    of the best way to store the cells and then we can go

     5    on from there.  Because otherwise, we are going to be

     6    trying to interrupt a death process that is more than

     7    likely not going to be apoptosis but some mechanical

     8    death due to pH, et cetera.

     9                So what next?  As I say, this is basically

    10    summarizing what I just said.  How representative is

    11    our source?  It looks like it is not very

    12    representative of what other people are getting. 

    13    Certainly not -- we have seen some of the data in

    14    terms of consistency historically that Susan Leitman

    15    has shown us and that is amazingly different and

    16    encouraging.  

    17                Very lastly, just a slide of who actually

    18    really did the work.  The work was actually done by

    19    Theresa, Steve, and Shannon in terms of the science

    20    and Karent Valentino and David Higgins, who collected

    21    a lot of the data and who are our clinical development

    22    people and got us in touch with everyone.  I sort of

    23    mainly drank coffee, discussed UNC basketball and

    24    provided amusing anecdotes.  Thank you.  

    25                CHAIRPERSON HARVATH:  Okay.  Dr. Ambruso?

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     1                DR. AMBRUSO:  I guess this microphone is

     2    on.  Dave, what kind of bags did you use to collect

     3    your samples in?

     4                DR. STRONCEK:  We collected them in

     5    originally the bags that come with the kit and then we

     6    transferred.  We tried in larger bags, some live cell

     7    bags, and teflon bags.  Live cell is a Baxter bag that

     8    is supposed to be more breathable, and it didn't seem

     9    to make much difference with storage.  We also tried

    10    teflon bags because we wanted the smaller bags, some

    11    with about a 30 or 40 ml capacity so we could aliquot

    12    these products and try some different storage

    13    conditions.  What was available was from a local

    14    company here that will manufacture teflon bags in

    15    about any size we wanted.  So that is why we picked

    16    those.

    17                DR. AMBRUSO:  These are not appreciably or

    18    don't allow enough gas exchange perhaps like

    19    platelets?

    20                DR. STRONCEK:  Well, the teflon bags are

    21    supposed to be extremely breathable.  They are

    22    supposed to be better than some of the other bags.

    23                DR. AMBRUSO:  You didn't see -- I mean one

    24    of the ways to approach this might be if you allowed,

    25    as with platelet storage, if you alloed CO2 to diffuse

                                                                        254

     1    out.  Maybe that might help with the pH.  

     2                DR. STRONCEK:  Yes.  That would be one of

     3    the things to try.

     4                AUDIENCE MEMBER:  Hi, Dave.  I don't know

     5    much about neutrophils, but if given a carbon source,

     6    will they take one that would go into the Krebs cycle

     7    rather than glycolysis?

     8                DR. STRONCEK:  I don't know. Do you mean

     9    what if we incubated them in a CO2 incubator at room

    10    temperature?

    11                DR. TORLINI:  Or acetate.  People are

    12    looking at platelets for acetate.  So you don't make

    13    an acid by-product.

    14                DR. STRONCEK:  That would probably be

    15    worth a try rather than trying to add or dilute out

    16    the products.

    17                AUDIENCE MEMBER:  Yes.  That is what I was

    18    thinking.

    19                CHAIRPERSON HARVATH:  Okay.  I would like

    20    to thank everyone who participated in this conference

    21    and all of those of you who have stayed here to the

    22    closing moments of the conference.  I hope our

    23    colleagues at NIH who were here to hear about the

    24    areas that are in need of research support in this

    25    area heard what all of you had to say.  I know that

                                                                        255

     1    one of the reasons for having this meeting transcribed

     2    was so that we could have a record of the current

     3    thinking in this area.  And also we look forward to,

     4    I think, continuing the dialogue and looking at the

     5    progress in this area, particularly those of you who

     6    have already discussed your intentions to pursue a

     7    multi-center type of study design to look at these

     8    basic issues.  I don't know if Dr. Snyder is still in

     9    the audience.  Is he here?  No.  Okay.  I would like

    10    to also thank him for his comments and input during

    11    the meeting as well and some of the suggestions that

    12    he had.  It has been a long couple of days.  We have

    13    heard a lot and I want to again thank all of you on

    14    behalf of the organizing committee and let you enjoy

    15    what is left of your Friday afternoon.

    16                Thank you.

    17                (Whereupon, at 3:41 p.m., the workshop was

    18    concluded.)

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