Hemopoietic Precursors in Human Bone Marrow Transplantation

Transcription

1 International Journal of Cell Cloning 4: Suppl 1 (1986) Hemopoietic Precursors in Human Bone Marrow Transplantation H.A. Messner Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada Key Words. Clonogenic precursors * CFU-gem * BFU-e CFU-m * CFU-c - Hemopoietic growth factors * Cell culture Cell cycle status Introduction The majority of bone marrow transplant recipients with long-term disease-free survival are usually able to produce peripheral blood cells in normal frequency. In spite of these normal values, transplant recipients appear to be at higher risk to suffer from severe life-threatening infections and display difficulties coping with increasing demands. This problem may relate to an incomplete reserve of early hernopoietic precursors within the engrafted marrow. We have, therefore, asked two questions: 1. Does the frequency of clonogenic precursors and their proliferative status return to normal values? 2. Does the time up to engraftrnent and the duration of survival correlate with the frequency of clonogenic cells in the transplant inoculum or can they be predicted by colony formation of the host prior to transplantation? Assay for Clonogenic Precursors The assays for clonogenic hemopoietic precursors are well established. Briefly, Percoll-separated mononuclear bone marrow cells of density less than g/ml are plated in methylcellulose supplemented with human plasma, Iscove s minimum essential medium, 2-mercaptoethanol and PHA-LCM as a source of hemopoietic Correspondence: Dr. Hans A. Messner, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K /86/$2.00/0 oalphamed Press, Inc.

2 Messner 12 growth-promoting factors [l, 21. Plates are scored after 14 days of incubation at 37T in humidified air, supplemented with 5% COz. These growth conditions support the development of multi- and single-lineage colonies, thus providing a measurement for pluripotent and committed hemopoietic precursors. In addition, the use of human plasma in the system has given the opportunity to assay plasma samples in transplant recipients for the presence of hemopoietic growth factors. These may be anticipated during early bone marrow regeneration. Hemopoietic Precursors in Bone Marrow Transplantation Recipients The culture analysis was performed on 103 bone marrow recipients and their respective donors. Almost all recipients demonstrated some colony growth one month after transplantation [3]. The mean values of clonogenic cells for the whole group may increase slightly over the subsequent two or three months and then remain constant up to the maximal follow-up of five years at 30%-40% of normal. These values are independent of the age, diagnosis and status of disease at transplantation. In spite of the decreased frequency of clonogenic precursors, normal peripheral blood parameters are maintained. This may be feasible by involving a greater than normal proportion of clonogenic precursors into ongoing proliferation. This hypothesis was tested by determining the number of clonogenic precursors in S-phase using the H3TdR suicide technique [4]. Incubation with H TdR of normal bone marrow samples prior to plating resulted in a reduction of the plating efficiency that ranged from 5% -35% of pluripotent precursors. In contrast, bone marrow transplant recipients whose counts had returned to normal appeared to be more sensitive to preincubation with H TdR. This observation is consistent with the view that hemopoiesis in bone marrow transplant recipients is maintained by a smaller number of precursors. All of these appear to cycle actively, thus indicating a decreased reserve of clonogenic precursors. Contribution of Cells in the Winsplant Inoculum to the Clinical Outcome Each one of the transplant inocula was collected with the target to provide 3 x 108 nucleated cells per kg of the recipient. An aliquot was used to enumerate the frequency of clonogenic precursors in culture. Considerable patient-to-patient heterogeneity was observed. The total number of nucleated cells varied as much as threefold covering a spread from 1.2 to 4.2 x 1Olo with a median of 2.4 x lolo cells. The frequency of clonogenic cells showed even greater variation of up to eightfold differences. The frequency of each of these subpopulations was exam-

3 Hemopoietic Precursors in BMT 13 ined to determine their influence on the clinical outcome measured by the time that had elapsed to achieve engraftment and by the length of survival. For this study, time to engraftment was defined as the interval required to sustain more than 50,000 platelets, 50,000 reticulocytes and 1,500 neutrophil granulocytes per mm3. Using a uni- and multivariate analysis, clonogenic precursors in the transplant inoculum were not correlated with either measurement of clinical outcome. In contrast, the total number of nucleated cells in the transplant inoculum contributed significantly to survival. Patients that had received a higher cell load had a higher probability of survival independent of other risk factors. This result was somewhat surprising. A number of hypotheses may be advanced to explain the findings. For instance, the clonogenic cells measured by the employed assays are not important for engraftment. This notion may be supported by observations in autologous bone marrow transplantation, where the transplanted marrow was pretreated with 4-hydroperoxycyclophosphamide. Functional grafts were obtained even after clonogenic cells were reduced to 10% or less [5]. It is conceivable that stable engraftment is the result of a more primitive stem cell represented in even lower frequency. A higher number of nucleated cells may then have a higher probability to contain them in sufficient frequency. Alternatively, engraftment may not only depend upon clonogenic precursors but also require the simultaneous transfusion of auxiliary cells, such as T lymphocytes. This possibility is supported by experiences transplanting T cell depleted bone marrow. Difficulties with engraftment are encountered in 1O%-l5% of these transplants. In a preliminary study, attempts were made to increase the frequency of nucleated cells in the transplant inoculum above the usual value of 3 XlOVkg recipient. Four of the five patients evaluated demonstrated clonogenic precursors as early as days 14 and 28 in a frequency that approximated normal values. The number of studied patients is too small and the time of follow-up is too short to determine whether the increased number of transplanted cells is not only reflected by an increased frequency in clonogenic precursors in the recipient but also by improved survival. Influence of Clinical Outcome by Clonogenic Precursors in the Recipient Prior to Transplantation Bone marrow samples from each transplant recipient were assessed prior to transplantation for the frequency of pluripotent and committed hemopoietic precursors. The influence of each of these values on clinical outcome was assessed using time to engraftment and survival as measurements.

4 Messner 14 The study revealed that only clonogenic precursors committed to megakayocytopoiesis (CFU-m) significantly influenced the time to engraftment. Recipients with high pre-transplant values of CFU-m engrafted faster than recipients with low CFU-m values. None of the pretransplant clonogenic precursors in the recipient had any influence on survival. The role that CFU-m in the recipient may play resulting in the observed correlation is open for speculation. It is unlikely that they reflect a more primitive stem cell population, although data are available in murine hemopoiesis that CFU-m and day 14 CFU-s [6] are recognized by the identical monoclonal antibody Qa-m2. Alternatively, the growth of CFU-m may reflect events in vivo that support the growth of clonogenic precursors. It may therefore indirectly measure the activity of auxilliary cell populations or the release of stimulatory molecules required for CFU-m growth. Based on this speculation, attempts were made to measure a growth supporting influence directly. CFU-m represent a suitable model since megakaryocyte colony formation is dependent upon the presence of a source of human plasma in the cultures [l, 21. Colony formation with normal human plasma, however, is only observed if a source of Meg-CSA is added. This can be provided by addition of PHA-EM. In contrast, plasma from patients with perturbed megakaryocytopoiesis, as for instance observed in aplastic anemia, may contain substances that not only support colony growth, but also replace the requirement for exogenous Meg-CSA. The question was asked whether or not similar activities can be observed in transplant recipients and whether these are predictive of engraftment [2]. Plasma samples were obtained before transplantation and serially thereafter. Routinely stimulatory activities can be identified as early as during administration of the ablative chemotherapy. In general, two main activity patterns were identified. Some patients increased the stimulatory activities to peak values between day 10 and day 20. Subsequently, these peak values decreased and disappeared approximately 30 days after transplantation. In a second subset of individuals, activities are sustained in plasma well beyond day 30. Attempts to correlate the activity within the plasma of transplant recipients yielded the following information. Recipients whose plasma returned to normal values within 30 days after transplantation tended to recover platelets faster than patients with sustained activity. Thus, delayed engraftment appears to be more related to cellular problems rather than the absence of stimulators. These observations provide some insight into the complexity of events that are associated with engraftment. In addition, one has to realize, however, that the interactions with drugs such as steroids and cyclosporine A, antiviral agents, the development of infections and GVHD may further complicate the issue. More!

5 Hemopoietic Precursors in BMT 15 detailed studies are required to delineate the influence of each of these nonhemopoietic parameters. References Messner HA, Jamal N, Izaguim C. The growth of large megakaryocyte colonies from human bone marrow. J Cell Physiol 1982;1: Solberg LA, Jamal N, Messner HA. Characterization of human megakaryocytic colony formation in human plasma. J Cell Physiol 1985;124: Messner HA, Curtis JE, Minden MD, et al. Clonogenic hemopoietic precursors in bone marrow transplantation. Submitted. Minden MD, Till JE, McCulloch EA. Proliferation state of blast cell progenitors in acute myeloblastic leukemia (AML). Blood 1978;52: Rowley SD, Stuart RK. 4-HydroperoxycycIophosphamide (4-HC) effects on human pluripotent stem cells (CFU-gemm) in vitro. J Exp Hematol 1983;Suppl 1: Abstract No. 11. Harris R4, Hogarth PM, Wadeson LJ, Collins P, McKenzie IFC, Penington DG. An antigenic difference between cells forming early and late haematopoietic spleen colonies (CFU-s). Nature 1984;307: Discussion Ho: I would just like to point out that the quality of cells that you are obtaining in the latter part of the operation is probably not the same as that you obtained in the first 15 minutes of aspiration. So, we re dealing with different populations of cells here. This complicates the type of analysis. I think it has been universally observed that there is no increase in graft-versus-host disease regardless of the total number of nucleated cells that one gives to the patient. That observation again complicates a comparison with T cell depleted marrow, I have one other comment in regards to interpreting these data on hemopoietic precursors. We have been puzzled by the same point that you have shown in regards to the clonogenicity of the marrow after transplantation. Just for the fun of it, we have discussed the suggestion among ourselves that perhaps stem cells are not as immortal as we used to think and that there may be a limit to the number of generations that these cells are able to go through. Probably these are lasting longer than the lifetime of an individual. However, when one uses these cells to transplant into a recipient, a number of generations have to be used in order to repopulate the bone marrow compartment. Therefore, these cells may then show this limit of clonogenicity after transplantation. Messner: You also find in the literature the experience that a reduction of the clonogenic components with agents like 4-hydroperoxycyclophosphamide has not im-

6 Messner 16 paired the ability of these marrow specimens to repopulate hemopoiesis. So that s precisely what I wanted to point out: that I think the available assays may therefore not measure the relevant population or we may not have the right concept of how these cells contribute to hemopoiesis. Sullivan: I presume these were patients with leukemia. Why was survival improved in those patients receiving greater than 2.4 x 10 0 nucleated cells in total? Was it protection from relapse, or lowered non-relapse death rates? Messner: The whole group of individuals were not selected for a specific disease. This group included some patients with aplastic anemia, and some patients with acute myeloid and lymphoid and chronic myeloid leukemia. The main problem that we encountered was related to infections and graft-versus-host disease. Our relapse rate in the whole group was relatively low, that is, 10% for patients transplanted in first remission of acute leukemia and patients with CML in chronic phase. Sullivan: We ve looked at this several times and several other centers have not been able to find such a correlate in leukemia patients receiving TBI and cell dose. Is it possible that the larger cell dose on that curve was a reflection of younger age recipients vis-a-vis less graft-versus-host disease and infections? Messner: That is a possibility. The data shown only considered patients below the age of 30. However, if the very same analysis is performed for older patients, the difference between patients receiving a low and high cell load is sustained. Sullivan: Yes, especially if you had to break it down under age 10, those children tend to get very large cell doses and that might influence the results. Messner: Our youngest patient in that group was 14, the majority between 18 and 30. Prchal: The speculation is possible that these clonogenic cells are restricted to a certain number of generations. But it is equally possible that two years posttransplantation, it was not possible to restore a normal stem cell pool of its usual size. Do you, or does anybody else, have any data at a later time than five years? Messner: We have reanalyzed the data after three, four and five years. About 15 patients have reached four years and about five have reached five years. No increment in clonogenic cells was observed in that small patient population.

7 Hemopoietic Precursors in BMT 17 Gelfand: This is a very impressive collection of data, but I wonder if in a way you don t set yourself an impossible task in that there are so many variables that go on in the post-transplant period, such as the influence of drugs, graft-versushost disease and so on. One of the impressive things that we have done with Me1 Freedman on colony formation is to study the impact of the lymphopoietic system on hematopoiesis by adding simple things such as exogenous IL2 to cultures. Since many of the post-bone marrow transplant recipients have problems with IL-2 production, including the number of T cells in IL2 production in their own culture system from the bone marrow with PHA, it seems to me that unless one is able to control these variables, it is almost impossible to get a correlation. Messner: The information that we can derive from these data is a quantitative and qualitative assessment of precursors at various timepoints. One can demonstrate a reduced frequency and one can show differences in their functional status. The chosen culture conditions provide IL-2-like substances in the form of PHA-LCM. Keating: Given the notion that perhaps the semisolid clonal assays are unable to assay for stem cells that cause engraftment, have you considered the use of longterm marrow cultures in assessing the effect of transplantation on marrow? Messner: Lmg-term bone marrow cultures may provide the opportunity to examine additional precursor populations that may be more relevant for transplantation. Keating: I was very intrigued by your recipient data with regard to time to engraftment with CFU-Meg. As you know, there is some evidence now from us and from other groups, that suggests that platelet-derived growth factor is a mitogen for the marrow microenvironment. The other piece of information is from Russell Ross s lab in Seattle that there is some evidence that megakaryocytic precursors synthesize PDGF in appreciable quantities. Given that data, do you think that the mechanism for the improved engraftment with increase in CFU-Meg in the recipient might be related to the increase in PDGF which might stimulate the newly generating microenvironment? Could you comment on that? Messner: This is quite possible. I m only aware of data where PDGF was used directly on bone marrow samples in the short-term assay. PDGF itself did apparently not directly influence the growth of clonogenic cells.

8 Messner 18 Keating: Both CFU-gem and also BFU-e have been shown, in marrow at least, to be augmented by PDGF. This probably functions through an accessory cell population. Saunders: I wonder how specific the phenomenon of low stem cell colonies posttransplant is. A number of years ago, I had the opportunity to do some follow-up marrows on patients with aplastic anemia who were not transplanted. There were just a few of them. They were, for all intents and purposes, spontaneous cures of aplastic anemia. They had normal blood counts. They were anywhere from 5-10 years post-aplasia. And they had very few stem cells in their marrows. Messner: I can only refer back to a number of patients with acute myeloid leukemia, who were examined three and four years after completion of chemotherapy. These patients also demonstrated very low frequency of clonogenic cells. A cell cycle state analysis, on the same samples, demonstrated increased cell cycle activity. So I think an injured marrow can really be reflected in the frequency of precursors and in their cell cycle state. Miller: The critical question in these low stem cells is whether one is looking at a small mismatch between host and donor or whether one is looking at an injured environment. Has anyone done the appropriate mouse experiment, which would be to look at mice that have been radiated and reconstituted with syngeneic bone marrow and then followed over a long period of time? That s not quite the same as the decline experiment, which does show serial decline in stem cells. Has that experiment been done? Messner: I have not seen any data. Gleichmann: I have a similar question. Your data remind me of some of the older findings in mice that were called allogeneic inhibition or Fl hybrid resistance, that describe the poor engraftment and rather small donor cell inoculum of stem cells that can rarely be overcome by increasing the donor cells. I wonder whether this has anything to do with that. Therefore, it would be interesting to control this in identical twin donor-recipient pairs, because there you should not see it. And that could also answer the question raised before, whether there is a poor proliferative capacity or restricted proliferative capacity of the stem cells or not, whether this is allogeneic inhibition. Messner: I don t have sufficient transplants in twins to be able to comment.