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             UNITED STATES OF AMERICA

           FOOD AND DRUG ADMINISTRATION

   CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

 

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

 

                   34^th MEETING

 

                     THURSDAY,

                 FEBRUARY 27, 2003

 

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

 

PRESENT:

 

DANIEL R. SALOMON, M.D.     Chairman

JONATHAN S. ALLAN, D.V.M.   Member

DAVID M. HARLAN, M.D.        Member

KATHERINE A. HIGH, M.D.     Member

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

JOANNE KURTZBERG, M.D.      Member

ALISON F. LAWTON            Industry Representative

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

RICHARD C. MULLIGAN, Ph.D.  Member

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

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

ANASTASIOS A. TSIATIS, Ph.D. Member

GAIL DAPOLITO               Executive Secretary

 

NIH REPRESENTATIVE

LIANE HARVATH, Ph.D.

 

GUEST SPEAKERS

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

PABLO RUBINSTEIN, M.D.

JOHN WAGNER, M.D.

 

PRESENT FROM FDA:

PHILIP NOGUCHI, M.D.

RUTH SOLOMON, M.D.

ELLEN LAZARUS, M.D.

STEPHEN LITWIN, M.D.

KAREN D. WEISS, M.D.

 

 

 

 

                     I-N-D-E-X

 

                                              Page

Morning Session

 

Welcome and Introductory Remarks                 4

      Daniel Salomon, M.D., Chair

 

Meeting Statement                                4

      Gail Dapolito, Executive Secretary

 

Presentation of Certificate of Appreciation     11

to Dr. Salomon

      Karen Weiss, M.D. &

      Philip Noguchi, M.D.

 

Topic I: Unrelated allogeneic hematopoietic stem/

      progenitor cells from placental/umbilical

      cord blood for hematopoietic reconstitution

 

      FDA Introduction                           

            Ellen Lazarus. M.D.                  15

            Stephen Litwin, M.D.                 20

 

      Guest Presentations

            John Wagner, M.D.                    45

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

            Question & Answer Period             87

            Pablo Rubinstein, M.D.              120

 

Open Public Hearing

 

      Mr. Steve Barsh                          159

      Mr. Glen Ackerman                        165

      Ms. Faraday Niakani                      167

      Mr. Joel Ruderman                        167

      Ms. Kathleen Kim                         169

      Mr. Herb Lee                             171

      Mr. Steven Sprague                       174

      Ms. Heidi Shaw-Tweten                    179

      Ms. Serls                                180

 

Afternoon Session

 

Committee Discussion of Questions              209

 

                 I-N-D-E-X (con't)

 

Topic II: Update CBER Research Programs       Page

 

      Division of Monoclonal Antibodies          

      Overview

            Keith Webber, Ph.D.                 326

 

      Laboratory of Cell Biology

            Kathy Clouse, Ph.D.                 329

            Ingrid Markovic, Ph.D.              335

            David Frucht, M.D.                  340

 

      Laboratory of Immunobiology

            Ezio Bonvini, M.D.                  346

 

      Laboratory of Molecular and

      Development Immunology

            Steven Kozlowski, M.D.              351

 

 

 

 

 

 

               P-R-O-C-E-E-D-I-N-G-S

                                         8:10 a.m.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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