Post Marrow Transplantation Research Articles

The Determinative Role of Hematopoietic Stem and Progenitor Cell Graft Composition on the Engraftment Kinetics after HSCT

The importance of hematopoietic stem and progenitor cell (HSPC) dose in the outcome of hematopoietic stem cell transplant (HSCT) has been demonstrated by analyses of the threshold dose of CD34+ cells required to achieve donor engraftment, usually defined as endpoints of donor-derived neutrophil (PMN) or platelet (PLT) absolute numbers, and RBC transfusion independence. We recently described the heterogeneous HSPC composition of prospectively obtained umbilical cord blood samples and lack of correlation between CD34+ cell dose and the frequency of hematopoietic stem cells (HSC)(Mantri S et al, Blood Adv 2020).We describe here a pilot analysis of the relationship between HSPC graft composition and engraftment after HSCT. The study population is comprised of 17 children (3.4-22 years, median 13 years) treated with αβT/CD19B - cell depleted mobilized peripheral blood stem cell (PBSC) HSCT (αβhaplo-HSCT). Eight patients had acute lymphoblastic leukemia, seven had acute myeloid leukemia, and two myelodysplastic syndrome. All patients received serotherapy with Thymoglobulin and Rituximab, and a myeloablative conditioning consisting of either TBI 1200 cGy in combination with Fludarabine/Thiotepa (15 patients), Melphalan/Thiotepa (1 patient), or TBI (400 cGy) with Fludarabine/Thiotepa (1 patient receiving a second HSCT). Only 2/17 patients received 1-2 doses of G-CSF before Day+30.The CD34+ Lin- cells in the PBSC products were analyzed for HSPC composition, including HSC (CD38- CD90+ CD45RA-), Multipotent Progenitors (MPP, CD38- CD90- CD45RA-), Common Myeloid Progenitors (CMP, CD38+ CD123+ CD45RA-), Granulocyte-Monocyte Progenitors (GMP, CD38+ CD123+ CD45RA+), Megakaryocyte-Erythroid Progenitors (MEP, CD38+ CD123- CD45RA-) and Common Lymphoid Progenitors (CLP, CD38+ CD127+). Similar HSPC analyses were performed on marrow obtained at Days +30, +60, and +90 post-HSCT. The CD34+ cell dose in the grafts ranged from 8.5-40 x 10e6/kg recipient body weight (mean 15.6). The median time to Absolute Neutrophil Count (ANC) > 500 or 1000/mm 3 were Days +12 and +14, respectively, and for PLT > 50K or 100K/mm 3 Days +14 and +15, respectively. The time to either PMN or PLT engraftment did not correlate with the HSC dose. In contrast, the GMP dose was predictive of PMN engraftment: 7/8 patients receiving the highest GMP dose achieved ANC > 500 at 0-3 days before the median day of engraftment, while PMN engraftment was delayed by 1-5 days beyond the median in 6 of the 9 receiving the lowest GMP dose (χ 2 = 8.14, p=0.004)(Fig A). Of the patients with the highest MEP dose, 7/8 achieved PLT >50K/mm 3 0-4 days before the median, while 5/9 receiving the lowest dose engrafted 1-6 days beyond the median (χ 2 4.9, p=0.026) (Fig B). In the first 100 days post-HSCT, naïve CD4+ T-cells were all CD31+ CD45RA+ Recent Thymic Emigrants (RTE), indicating that they were newly produced T cells and not adoptively transferred from the αβhaplo-HSCT. The HSC dose did not correlate with the number of naïve CD4+ T cells until Day+180 (Fig C). Like PMN and PLT recovery, early T lymphoid recovery after HSCT was mainly derived from infused CLP, and likely switched to HSC-derived lymphopoiesis by Day +180. Consistent with this concept, HSC dose in the PBSC products was unrelated to the numbers of committed progenitors, e.g., CMP, MEP, CLP, in early (Day +30) post-transplant marrow samples.These data are consistent with clonal analyses of patients receiving lentiviral gene therapy and murine experiments demonstrating prolonged steady-state contribution of committed progenitors to peripheral blood cell maintenance (Biasco L et al, Cell Stem Cell 2016; Sun J et al, Nature 2014). While post-HSCT PMN or PLT numbers are frequently equated with “HSC engraftment” and naïve T-cell numbers with HSC-derived immune reconstitution, these early events reflect blood cell production by committed progenitors, which are variably present in the grafts. Although CD34+ cell dose is currently used to predict post-transplant engraftment and to qualify stem cell products for release, more accurate clinical predictions may be determined by the HSPC grafts' composition. Further, engineering of the progenitor composition of clinical HSCT products, e.g., co-transplantation of additional committed progenitors like GMP or CLP, could be strategically used to control post-transplant lymphohematopoietic recovery. [Display omitted] DisclosuresParkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

32 Laboratory Evaluation for Therapy-Related Myeloid Neoplasia-Associated Changes After Autotransplant for Multiple Myeloma

Therapy-related myeloid neoplasms (tMNs), especially MDS and AML, are well-known, serious complications of chemotherapy agents, such as topoisomerase II inhibitors and alkylating agents. It is less clear if newer efficacious agents such a lenalidomide (LEN) carry an increased risk of tMN. The overall incidence of tMNs in treated multiple myeloma (MM) patients is approximately 3%. Our laboratory evaluates a high volume of MM bone marrow (BM) biopsies with a full complement of ancillary studies, including many from patients that are post-autotransplant. To better understand the ordering pattern and utility of complex ancillary studies to detect tMN, we queried the departmental pathology database from May 2011 to December 2016 and found results for 1,212 MM patients who received biopsies following autotransplantation. We identified patients that had MDS fluorescence in situ hybridization (FISH) abnormalities, myeloid-related karyotypic abnormalities, or abnormal myeloblasts by flow cytometry as indicators of possible tMN, excluding MM-associated changes. We found two of 14 patients had an abnormal MDS FISH panel result and a third had a borderline/equivocal result. Myeloid-related karyotype abnormalities were observed for 51/1,208 patients and defined the same abnormalities for the two patients with abnormal MDS FISH. These karyotype abnormalities were grouped into risk categories (18% high risk, 2% intermediate risk, 47% low risk, 33% uncertain risk). Seven of 1,212 patients displayed abnormal myeloblasts by flow cytometry, for which three patients also had an abnormal myeloid-related karyotype that fell into the high-risk group. A subset of these data was analyzed to test whether patients given LEN display increased tMN incidence. Three hundred ninety-seven patients received uniform pretransplant induction therapy with subsets receiving LEN-only maintenance or observation. No significant differences were found between groups concerning age, gender, ethnicity, Ig isotype, number of posttransplant marrow analyses, or average interval from transplant to last biopsy. Lab indicators of possible tMN for LEN-only (n = 13/271) and the observation group (3/92) were not statistically significant between groups (P = .77). Our data suggest several interesting conclusions. First, tMN-associated findings are rare in our patient population and consistent with reported rates. Second, from a lab perspective, MDS FISH provides little added value over routine karyotyping in evaluation for tMN, arguing against the use of this expensive test in this clinical situation. Third, while some published data suggest a higher rate of second malignancies in patients’ receiving LEN maintenance treatment, in our well-controlled subset analysis, LEN-only maintenance therapy does not yield significantly higher rates of tMN-associated lab findings.

Impending relapse of myelodysplastic syndrome after allogeneic transplant is difficult to diagnose and requires a multi-modal approach

BackgroundThe only potentially curative therapy for myelodysplastic syndrome is allogeneic hematopoietic cell transplant; unfortunately, there is a high relapse rate. The objective of this study was to perform a detailed clinicopathologic study of patients with relapsed myeloid neoplasm following allogeneic hematopoietic cell transplant for myelodysplastic syndrome.MethodsPre-transplant, post-transplant, and relapse bone marrow and peripheral blood morphologic features (including dysplasia) were retrospectively evaluated by study authors. Clinical features and results of cytogenetic analysis and engraftment/chimerism studies were obtained from the medical record.ResultsOur study describes 21 patients with a median time to relapse of 6 months (range 2–82). Ten of the patients relapsed with higher grade disease, including six with overt acute myeloid leukemia. Pre-transplant megakaryocyte dysplasia was associated with dysplastic megakaryocytes in the relapse specimen; however, neither erythroid dysplasia nor granulocytic dysplasia were associated with their counterpart in the relapse specimen. Relapse specimens had a lower marrow cellularity and higher blast percentage than pre-transplant disease. Cytogenetic comparisons before and after transplant showed variety, including clonal evolution (22%), the same abnormal clone (33%), or a different abnormal clone (22%).ConclusionsOur detailed review of post-transplant marrow biopsies prior to relapse highlights the difficulty in diagnosing relapse and particularly impending relapse.

Morphologic Examination of Sequential Bone Marrow Biopsies after Nonmyeloablative Stem Cell Transplantation Complements Molecular Studies of Donor Engraftment

Nonmyeloablative stem cell transplantation (NMSCT) is a mode of immunotherapy increasingly employed in treating hematologic, lymphoid, and solid tumors. Patients are monitored principally by molecular analysis of donor engraftment. To determine the role of morphologic examination of bone marrow after NMSCT. Seventy-three patients undergoing NMSCT under the Campath 1H (humanized anti-CD52 antibody) protocol were studied. Pretransplant and sequential posttransplant bone marrow specimens were evaluated and the findings were correlated with corresponding engraftment data. Pretransplant bone marrow specimens from 43% of the patients were involved by disease, and these marrow specimens were significantly more cellular than those that were free of disease. Morphologically detectable disease was still present in day 14 posttransplant marrow specimens in more than one half of these patients, but there was no difference in engraftment in those with or without marrow disease. Early posttransplant marrow in nearly one half of the patients showed myeloid hyperplasia and atypical localization of immature myeloid precursors. Marrow cellularity for the first 2 months after NMSCT was significantly lower in those patients receiving stem cells mismatched at 1 to 3 loci as compared with those who received fully matched grafts (mean cellularity, 38.1% vs 54.1% at day 14). Marrow failure without recurrent disease at 3 to 6 months after transplant was detected by engraftment study in only approximately 15% of cases. Similarly, early recurrence of disease was detected first by morphologic examination in 4 of 13 cases before a decline in donor engraftment occurred. Morphologic examination of bone marrow provides additional information that is complementary to donor engraftment analysis for optimal management after NMSCT.

Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graft-versus-host disease risk

L-leucyl-L-leucine methyl ester (LLME) prevents GVHD in several animal models by depleting dipeptidyl peptidase I (DPPI)-expressing cytotoxic cellular subsets. However, clinical application has been hampered by difficulties in stem cell engraftment following treatment of donor bone marrow inocula with LLME at the concentrations necessary to purge DPPI-expressing T-cells. Noting that T-cells can mediate graft-versus-leukemia (GVL) responses via both perforin (usually co-expressed in cytotoxic granules with DPPI) and Fas ligand pathways in a murine model, we hypothesized that LLME might be useful for treatment of delayed DLIs for potential GVL activity with a decreased risk of GVHD induction. In regard to the clinical setting, the ex vivo use of LLME for this purpose would circumvent any toxicity issues for donor stem cells, because by that time patients would have already achieved successful engraftment. For our preclinical studies, we used the haploidentical C57BL/6 (B6) (H2b) --> ((B6 x DBA/2)F1 (H(2b/d)) murine model with lethally irradiated hosts that had received transplants of T-cell-depleted bone marrow cells and were challenged with the MMD2-8 myeloid leukemia line (H2d) of DBA/2 origin. A DLI of LLME-treated donor splenocytes, from B6 mice presensitized to recipient alloantigens, was administered in varying doses 14 days post-marrow transplantation, and the potential for both GVHD and GVL activity was assessed. All mice that received any dose of LLME-treated DLI survived indefinitely, without evidence of cachexia nor B-cell hypoplasia, in contrast to the severe and lethal GVHD induced by mock-treated DLI. Histological analysis largely correlated with the symptomatic findings and revealed no GVHD-like lesions in the spleens of LLME-treated DLI recipients, although some mice displayed various degrees of hepatic mononuclear infiltration. Most notably, mice given LLME-treated DLI also experienced DLI dose-dependent increases in survival against the challenge with the MMD2-8 leukemia. LLME-treated splenocytes remained immunocompetent, as these cells could proliferate in response to mitogens and to restimulation with ovalbumin when used as a recall antigen. In conclusion, LLME-treated DLI possesses immune potential and, in particular, GVL activity without inducing clinically evident GVHD.

The effect of donor leukocyte infusions on development of EBV specific immunity post-marrow transplantation

The effect of donor leukocyte infusions on development of EBV specific immunity post-marrow transplantation

The graft-vs.-leukemia effect. Implications for post-marrow transplant antileukemia treatment.

Over the past 30 years, evidence supporting the existence of an antileukemic effect of allogeneic bone marrow has rapidly expanded. Early animal studies demonstrated this "graft-vs.-leukemia" (GVL) effect in association with graft-vs.-host disease (GVHD). Clinical evidence for the GVL effect derived from experience in allogeneic bone marrow transplantation (BMT) includes the following: the association of GVHD, both acute and chronic, with a decreased rate of leukemic relapse, the occurrence of a higher relapse rate following identical twin transplants compared to allogeneic major histocompatibility complex-matched sibling transplants, the decrease of GVHD and increase of leukemic relapses with T-cell depletion of donor bone marrow, and that allogeneic marrow transplants without GVHD have a lower leukemic relapse rate compared to identical twin transplants and T-cell depleted transplants. In addition, some reports indicate that modifying the immunosuppressive regimens (consisting of cyclosporine A or methotrexate) that are aimed at prevention of GVHD can affect leukemic relapse rates. Although the mechanism of this GVL effect is poorly understood, there are several favored hypotheses. Cytotoxic T cells, which may mediate GVH effects, could also mediate a GVL effect. There may also be T cells with specificity for destroying leukemic blasts alone. The natural killer/lymphokine-activated killer cytotoxic cell may play a role in the GVL effect. These nonspecific cytotoxic cells can be activated with cytokines induced in the transplant setting. Finally, the GVL effect could be secondary to the in vivo induction of various cytokines that either have direct antileukemic effects or that induce leukemic differentiation. Advances in our basic understanding of the immune system and its activation, and the availability of purified immunoregulatory cytokines, have revealed new ways to induce GVL-like effects. Promising results with therapy based on use of interleukin-2 in solid tumors have been seen. It is hoped that these advances will enable the "antileukemic" components of the GVL effect to be prospectively controlled (as part of the technique of marrow transplant) and intentionally used as leukemic therapy in patients undergoing bone marrow transplantation.

Human Herpesvirus-6 (HHV-6) Infection in Allogeneic Bone Marrow Transplant Recipients: Evidence of a Marrow-Suppressive Role for HHV-6 In Vivo

Sixteen adults were studied for the first 100 days after allogeneic bone marrow transplant to assess the pathogenic role of human herpesvirus-6 (HHV-6) infection in patients with unexplained febrile illnesses. HHV-6 was directly isolated from the blood of 6 patients. Analysis of the clinical courses of these 16 patients revealed otherwise unexplained posttransplant marrow suppression in 5 patients. Idiopathic marrow suppression occurred more frequently in patients with concurrent HHV-6 viremia (4/6) than in those from whom HHV-6 was not isolated from peripheral blood (1/10, P < .05). An etiologic role for the virus was also supported by isolation of HHV-6 from the bone marrow of all 4 patients at the time of marrow suppression and by in vitro colony-forming unit (cfu) assays that demonstrated that HHV-6 could inhibit cfu-granulocyte-macrophage and burst-forming unit-erythroid growth from human bone marrow. By restriction enzyme mapping, all clinical isolates were type B, suggesting that bone marrow transplant recipients may be preferentially infected with and reactivate this HHV-6 subtype. This study implicates HHV-6 as a novel cause of bone marrow suppression in marrow transplant recipients.

Mechanisms in Immune Recovery After Bone Marrow Transplantation

Bone marrow transplantation usually entails the ablation of the recipient's immune system. The recovery of immunity post-marrow transplantation is a complex process dependent on a number of pre- and posttransplant factors. This knowledge has led to the development of novel approaches to supportive care of the patient, as well as to methods of augmenting immunity. Hematopoietic growth factors are now used to speed neutrophil recovery. Certain lymphokines are being investigated for the enhancement of cell-mediated immunity, and gamma globulin preparations are used to support the deficiency in humoral immunity. This review summarizes the basic concepts of immune reconstitution posttransplant and discusses some of the therapeutic interventions that have been investigated. In addition, some general recommendations are offered regarding supportive care.

Developmental hierarchy during early human B-cell ontogeny after autologous bone marrow transplantation using autografts depleted of CD19+ B-cell precursors by an anti-CD19 pan-B-cell immunotoxin containing pokeweed antiviral protein

Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post-BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time-dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor-depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.

Developmental Hierarchy During Early Human B-Cell Ontogeny After Autologous Bone Marrow Transplantation Using Autografts Depleted of CD19+B-Cell Precursors by an Anti-CD19 Pan-B-Cell Immunotoxin Containing Pokeweed Antiviral Protein

Abstract Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post- BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time- dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor- depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.

Pulmonary Complications Following Bone Marrow Transplantation

Pulmonary complications are a common cause of morbidity and mortality after bone marrow transplantation. Some of these complications are infectious and related to the degree of ongoing immunosuppression, and thus vary with the posttransplant marrow reconstitution and the need for continued immunomodulating drugs. Noninfectious complications are related to previous chemotherapy, the transplantation protocol and conditioning regimen, and, in allogeneic transplants, the presence of graft-versus-host disease. The various complications are classified based on their timing after the transplant procedure. A diagnostic and therapeutic approach is outlined and the prognosis is discussed.

Flow cytometric analysis of circulating lymphocytes following human marrow transplantation.

Study of patients following marrow transplantation has revealed a number of phenotypically unusual cell types that appear to have unique functional correlates in vitro and possible unique clinical correlates in vivo. These include unusual T lymphocytes, such as the Leu4+ Leu2+ but Leu1- T cell; unusual NK cells, such as the Leu2+ Leu11+ NK cell; and unusual B lymphocytes, such as the Leu1+ B cell. Study of these cells both qualitatively to determine further aspects of their structure and function and quantitatively to determine their pattern of normal temporal reconstitution and how that varies under differing clinical conditions should continue to provide improved prognostic and diagnostic testing of postmarrow transplant patients. Furthermore, future study may help to provide information of use in designing improved therapeutic approaches.

Human bone marrow allograft recipients: production of, and responsiveness to, interleukin 2.

The production and utilization of interleukin 2 (IL 2) by peripheral blood mononuclear cells (PBMC) from 14 bone marrow allograft recipients was examined. PBMC from all patients were impaired in their ability to produce IL 2 when stimulated by phytohemagglutinin (PHA). On the average, the IL 2 activity produced by patients' PBMC was 8% of that from normal donors' PBMC. To examine the basis for this impaired T cell function in marrow recipients, the ability of a resting T lymphocyte population isolated from PBMC to respond to PHA and exogenously supplied IL 2 was analyzed. The proliferative response of resting T cells to PHA and IL 2, although low at early times post-transplant, reached near normal levels by 8 mo. Only two of 11 patients had normal numbers of precursor T cells that could respond. For all other patients the average number of precursor T cells was 10-fold lower than the average determined for normal donors. The impaired production of IL 2 by patients' PBMC may be due to this low precursor frequency. For some patients the rate and/or extent of clonal expansion of activated T cells appears to be greater than that of normal donors. The data suggest that the therapeutic application of IL 2 to such patients is unlikely to be successful in overcoming defects of T cell function before 8 mo post-marrow transplant.

Morphologic aspects of bone marrow transplantation in patients with aplastic anemia

Pre- and post-transplant bone marrow samples from 20 patients with aplastic anemia were studied. Morphologic evidence of marrow reconstitution was noted in 18 patients one to three weeks following transplantation. In most instances the engrafted marrow elements in early weeks appeared as small clusters of erythroid or myeloid precursors. Bone marrow biopsy or clot sections obtained four to eight weeks after transplantation were more cellular with larger clusters of hematopoietic cells, which were most often composed of mixed cellular elements, including megakaryocytes. Two patients with morphologic evidence of engraftment died shortly after transplantation and were excluded from further analysis. In four of the remaining 16 patients grafts were “rejected” three to eight weeks after transplant. A fifth patient who received a marrow graft from his identical twin showed a transient increase in marrow cellularity without clinical improvement. However, the second marrow transplantation in this patient using the same donor after conditioning with cyclophosphamide resulted in moderate clinical improvement. In four of the five patients developing graft “rejection” or failure there was an increase in marrow mast cells in pre- and post-transplant marrow samples. In contrast, only two of 11 patients with successful engraftment had an increase in mast cells. Although the pathophysiologic role of mast cells in marrow transplantation is unclear, the present study suggests a possible inverse correlation between the numbers of marrow mast cells and the likelihood of successful engraftment. Bone marrow samples in patients with graft versus host disease displayed a slight increase in the number of eosinophils, lymphocytes, and plasma cells.