Cord Blood Stem Cell Banking and Transplantation JOHN W. ADAMSON New York Blood Center, New York, New York, USA Key Words. Cord blood Stem cells Cord blood banking Cord blood transplantation. Cord blood.stern cell 6iology. Graft-versus-host disease Graft-versus-leukemia effect. Cell dose threshold ABSTRACT Cord blood banking for the purpose of stem cell transplantation is a rapidly growing area of medical interest. Based on the fact that cord blood contains large numbers of stem and progenitor cells, transplantation of cord blood for marrow reconstitution was first attempted in 1988. The success of this initial transplant between related donor and patient rapidly led to the establishment of efforts to collect, store and eventually transplant unrelated cord blood samples. A collection and storage program established by the New York Blood Center has led to more than 400 such transplants. The results demonstrate acceptable rates of engraftment and little graft-versushost disease compared to the results employing adult marrow. As a consequence of these observations, considerable effort is being made to establish cord blood banks around the world. Stem Cells 1997;1S(suppl 1):S7-61 THE NATIONAL MARROW DONOR PROGRAM Conservatively, there are at least 5,000 bone marrow transplants which cannot be performed in the United States each year because there is no matched donor available. To support patients in need, a National Bone Marrow Donor Program was established a number of years ago. Made up of volunteers who are willing to donate marrow for unrelated recipients, the Program (and its Registry) provides hope for those who lack a suitably matched donor. However, the current need for transplant donors is fulfilled only partially by the Program despite the fact that each year there is an increase in the number of unrelated bone marrow transplants using donors identified by the Registry. There are limitations with the Registry; for example, ethnic minorities are underrepresented leading to redundant HLA types. Consequently, with 1 x lo6 individuals in the Registry, there is approximately a 30% chance of finding a donor who is fully HLA (six out of six antigens in common) compatible. If the number of potential donors is increased to 10 x lo6, there is a 40% chance of finding a fully compatible donor. As the number of potential donors increases, however, there is more redundancy in the common HLA types, and the number of new types increases only incrementally. Evidence of this is that only about 17% of African-American patients for whom a formal search of the Registry is requested will go on to receive a transplant, compared to 32% for Caucasian patients. The length of time to complete a search for a matched donor is also critical. Currently, the average time to complete a search is greater than four months. Patients with chronic disease or a malignant disease in stable remission can tolerate a delay in finding a donor, especially patients with chronic myelogenous Hematopoietic Stem Cells. STEM CELLS 1997;15(suppl 1):57-61 0 1997 AlphaMed Press. All rights reserved.
58 Cord Blood Banking and Transplantation leukemia or acute lymphoblastic leukemia. Other patients with less stable disease may not be able to wait to find a suitable and willing donor. Despite such limitations, the Program has made many contributions: it allows for the accumulation of a large number of potential marrow donors, it has been expanded worldwide and it provides hope for those patients without a related donor. Thus far, the Program has provided donors for more than 5,000 transplants in the United States. CORD BLOOD AS A SOURCE OF TRANSPLANTABLE STEM CELLS Over a decade ago, a series of studies revealed that cord blood is an abundant source of stem and progenitor cells and that placental/umbilical cord blood cells might be used for transplantation [ 1, 21. Quantitatively, cord blood contains high concentrations of progenitor cells which average 26,0001ml. This is a much higher concentration than found in normal adult peripheral blood and is a concentration which is as great as the concentration of progenitor cells mobilized by cytokines and/or chemotherapy. There are also differences in the proliferative capacity between progenitor cell types in cord blood and adult bone marrow. In fetal blood, about one-quarter of the total number of colony-forming cells (CFCs) will be mixed-cell CFCs. In adult marrow the percentage of mixed-cell CFCs is only 2%-3%. In addition, cord blood CFCs give rise to much larger colonies in vitro and there is a greater concentration of long-term culture-initiating cells in cord blood than in adult marrow. Finally, from a practical point of view, collection of cord blood cells offers no risk to the donor, ethnic diversity can be achieved, infectious diseases screened for and, once frozen, the cells remain viable for years. In addition, the cells are immediately available for transplantation. The first case which proved the principle of the ability of cord blood stem cells to reconstitute marrow function was that of a young man with Fanconi s anemia who was transplanted successfully using HLAmatched cord blood from an unaffected sibling [3]. The transplantation was carried out in 1988 and now, nine years later, the child remains well and hematologically normal. This initial positive result, along with substantial supporting laboratory data, led to the establishment of an International Cord Blood Transplant Registry which was designed to collect all available outcomes on cord blood transplants [4]. The first comprehensive summary of that effort was published in 1995. In that report Wagner et nl. summarized findings on 44 children (age range 0.8 to 16 years; median 4 years) who received sibling donor transplants [S]. Only 34/44 were HLA-identical. In evaluable patients, the median time to an ANC SOO/pl was 22 days (range: 12 to 46 days) and median time to a platelet count of 5O,OOO/p1 was 49 days (range: 15 to 117 days). Four patients did not engraft. In patients receiving mismatched cord blood transplants, graft-versus-host-disease (GVHD) was less severe than expected. Thus, cord blood cells appeared to be better tolerated by (and to better tolerate) a foreign environment. Beginning in 1993, the New York Blood Center initiated a program under the direction of Dr. Pablo Rubinstein to test the feasibility of a cord blood stem cell bank to collect, store, type and freeze up to lo4 cord blood samples and make them available for transplantation [6]. Important outcome variables included the number of stem cells needed to engraft an adult, a possible reduced graft-verus-leukemia effect (because of the reduced GVHD), and the existence of HLA blanks. As of January 1, 1997, there have been more than 3,000 requests for matches and over 300 unrelated transplants have been carried out using material provided by the New York Blood Center program. As with the results reported for the first related cord blood transplant, it is encouraging to note that the second patient to receive an unrelated transplant successfully engrafted and is alive and well three and one-half years later. In order to permit efficient storage of cord blood samples, Rubinstein and coworkers devised a closed system permitting volume reduction to a final storage volume of 25 ml [7]. This material has been used successfully in unrelated transplants and, coupled with a thawing technique which eliminates dimethylsulfoxide from the graft, allows speedier neutrophil recovery [S].
Adamson 59 As unrelated cord blood transplantation grows, a number of conclusions have emerged. First, there is clearly less GVHD than would be expected from an unrelated transplant employing adult marrow. This includes both acute GVHD and, particularly, chronic GVHD. This permits transplantation with one or even two antigen mismatched cord blood stem cell preparations. Second, the time to platelet recovery is delayed following cord blood transplantation with a median time of 52 days to reach a platelet count of 20,0OO/p1. The time to neutrophil recovery (ANC of 500) is 23 days. It is not clear if post-transplant administration of hematopoietic growth factors such as G-CSF or the newly identified thrombopoietin will accelerate marrow recovery. Third, there is a relationship between cell dose and success of engraftment, although the reasons for the relationship are unclear. Finally, although adults have been successfully transplanted [9], it is not yet clear if this can be achieved routinely or if cell dose and degree of HLA disparity will be considerations in selecting the most appropriate graft. In summary, cord blood banking and the use of cord blood for purposes of marrow reconstitution have moved rapidly from theory to active practice. The encouraging early results should accelerate even more the exploration of this form of therapy. REFERENCES Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stendprogenitor cells. Proc Natl Acad Sci USA 1989;86:3828-3832. Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992;89:4109-4113. Gluchan E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-1178. Gluckman E, Wagner J, Hows J et al. Cord blood banking for hematopoietic stem cell transplantation: an international cord blood transplant registry. Bone Marrow Transplant 1992;ll: 199-200. 5 Wagner JE, Kernan NA, Steinbuch M et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346:2 14-2 19. 6 Rubinstein P, Rosenfield RE, Adamson JW et al. Stored placental blood for unrelated bone marrow reconstitution. Blood 1993;8 1 : 1679-1690. 7 Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placentavumbilical cord blood for unrelated hone marrow reconstitution. Proc Natl Acad Sci USA 1995;90:10119-10122. 8 Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157-166. 9 Laporte J-P, Gorin N-C, Rubinstein P et al. Cordblood transplantation from an unrelated donor in an adult with chronic myelogenous leukemia. N Engl J Med 1996;335:167-170. DISCUSSION Dr. Spangrude: My understanding is in the cord blood transplants, if one does a marrow biopsy you actually see lots of megakaryocytopoiesis in the marrow biopsies and so something is breaking down between that and the release of platelets in the blood stream. Is that right? Dr. Adarnson: I don t know that, but certainly the marrows are more cellular than one would anticipate based on the peripheral counts. That has been an observation that has been reported anecdotally. Dr. Torok-Storb: When the statement is made that there is no correlation between progenitor numbers, CD34 number, and engraftment kinetics with this product, is this analysis based on the pre-freeze determinations? Dr. Adarnson: Yes. Dr. Torok-Storb: That s what I couldn t figure out; whether they were doing the analysis on the actual product that was being infused or on pre-freeze numbers.
60 Cord Blood Banking and Transplantation Dr. Adamson: It depends on who s doing the recording. Dr. Torok-Storb: This could really impact on establishing the correlation and I have yet to see that information made completely available. Dr. Adamson: The point is well taken. Hal Broxrneyer probably knows more about freezing and thawing cord blood samples than anyone. There is a process now in place for manipulating cord blood samples in a closed system. This was developed by Rubinstein and others at the NY Blood Center and published last November in PNAS. So the process is there and it allows you to process cord blood samples, reduce the volumes to no more than 25 ml so the transplants that are being carried out are being camed out with frozen samples that are 25 ml in volume. With this processing there is full recovery of progenitor cell numbers. There are enough unrelated transplants that have been supported by this process such that you can make correlations based on the number of progenitor cells with confidence. They are pre-freeze progenitor cell assays, but enough work has gone into looking at the recovery of progenitor cell numbers before and after freezing to allow us with some confidence to think the correlation should hold. Dr. Torok-Storb: Well, the post-freeze numbers are done at a different institute. They re done at the institute where the transplant is being done. Dr. Adamson: Correct Dr. Torok-Storb: They have to be. And so the product is quantitated where it is harvested and frozen and then it s shipped, which is another variable. And then the institute that is doing the transplant does the second analysis, which could be an entirely different assay. Dr. Adamson: It could be an entirely different assay. Dr. Broxmeyer: Someday the world will get together and everybody will be willing to put their results into a registry, which is the way it started out. Everybody was happy to do that until everybody started becoming very possessive of their data. I think we ll know the answer to that. And right now, they don t know the lower limits yet that you need in order to be sure you re going to get successful transplants. Dr. Adamson: A single center at Duke has done over 45 unrelated transplants and so there is single center data coming along now with sufficient numbers to look at correlations. And using the data from that center, there is still no correlation yet between cell or progenitor cell dose and time to engraftment. Dr. Griffin: What was your frequency of engraftment? Dr. Adamson: Out of the first 106 patients that were supported by our program, 90 were evaluable for engraftment, which means alive at day 14, and 80 engrafted. Dr. Torok-Storb: Well, we ve found the lower limits in five patients. We ve transplanted five patients and none of them have survived. One patient died at about day 17 with an empty marrow. Dr. Quesenberry: Were those patients all adults? Dr. Torok-Storb: Yes. But the one thing that I can t figure out is the actual success rate for all patients treated with cord blood. It appears that if patients die early, they are taken out of the denominator. This makes it really difficult to evaluate this procedure. Dr. Broxmeyer: Is there any information yet that will relate the time to recovery to the HLA mismatch? Apparently, a lot of unrelated transplants have not been complete HLA matches. How will anybody else find out if the degree of mismatch relates at all to the time to engraftment? Dr. Adamson: I don t know whether anyone has looked at that data yet, so I can t tell you. Dr. Fibbe: Ths is a very important aspect, probably. I just like to remind you of the paper of Yair Reisner in Nature Medicine, (1995;1:1268-1278). It has shown that you can overcome HLA barriers by transplanting massive doses of T cell depleted stem cells. And this is in line with that. The higher degree of mismatch, the higher the number of stem cells you need. Dr. Broxmeyer: I don t think anybody has seen anything like that yet, but I don t know who did it or if the data have been accurately analyzed. Dr. Adamson: Well, this is obviously going to be a very important area and, as I say, the interest is sufficiently high that the NIH is going to expand collection activities substantially within the next year, as well as also transplant
Adamson 61 activities. Part of the problem is that the program has grown so fast that there are a lot of unpublished data that need to be analyzed and made available. Dr. Broxmeyer: What s the graft failure rate for bone marrow now? Dr. Torok-Storb: Graft failure or poor marrow function occurs in about 25% in an allogeneic setting based on an analysis of 487 allogeneic transplants done between January 1994-1995 Eight percent to 14% of those are early failures. These patients never engrafted. And the difference is dependent upon their HLA disparity. So 8% for related sibs, 14% for unrelated and mismatches. Of the ones that did engraft, so they reached an ANC of I,OOO/microliter at least, another 11% failed or experienced poor marrow function late. Dr. Kanz: What is the selection of the patients? This rate is very high. Dr. Torok-Storb: Well, you how Seattle does hard patients. We do all the patients nobody else wants to do. But these are our results. The early failures are primarily rejections. So that is not a stem cell problem. There are about I I% that fail late and by late I mean after day 120. They are not failures upfront. They engraft, they look okay, then they lose it. And these, too, are not stem cell problems. I ve studied these patients extensively and almost uniformly they can be attnbuted to complications in the microenvironment, either virus-mediated failures of the microenvironment or allogeneic reactions against the microenvironment. The most recent statistic that came out was an analysis of 1,500 allogeneic transplants in our long-term follow-up office. And that covers patients who are out 20 years. So they ve left our system. In this group there is a minimum of 8% late failures based on criteria that the patients die and the proceeding ANC counts were below 500. The statistician insists this is an underestimation because of our inability to follow up. Dr. Sharkis: You have no way of knowing whether the stem cells had anything to do with it. Dr. Torok-Storb: No, we just uncovered this statistic recently because we were preparing for a review and it surprised everybody. Dr. Hoffman: In the first group, do you attempt second transplants? Dr. Torok-Storb: In a few of them we attempt a second transplant. Some of them take, a lot of them don t take Dr. Papayannopoulou: Could some of these late failures be a myelodysplasia? Dr. Torok-Storb: Well, the patients are followed, so if it s a recurrent disease or if it s MDS or if it s a new disease, they usually pick that up. Such patients are not included in this number. Dr. Hoffman: How many of these 8% die of empty marrows? Dr. Torok-Storb: We don t know that because we don t marrow these patients. We re relying on the hometown doctors to make the diagnosis. These are patients that are not identified as MDS or relapses. So we specifically went out and looked for failures defined as an ANC less than 500 and death related to a low ANC that was not attributed to other causes. Dr. Quesenberry: Is there a correlation of the really late failures with the number of engrafted stem cells? Dr. Torok-Storb: I don t know. I would be surprised since we re giving so many cells, far more cells than patients need to engraft.