Jill Hows

Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not

Summary. In postnatal life, mesenchymal stem cells (MSC) self‐replicate, proliferate and differentiate into mesenchymal tissues, including bone, fat, tendon, muscle and bone marrow (BM) stroma. Possible clinical applications for MSC in stem cell transplantation have been proposed. We have evaluated the frequency, phenotype and differentiation potential of MSC in adult BM, cord blood (CB) and peripheral blood stem cell collections (PBSC). During culture, BM MSC proliferated to confluence in 10–14 d, maintaining a stable non‐haemopoietic phenotype, HLA class‐1+, CD29+, CD44+, CD90+, CD45–, CD34– and CD14 through subsequent passages. Using the colony forming unit fibroblasts assay, the estimated frequency of MSC in the BM nucleated cell population was 1 in 3·4 × 104 cells. Both adipogenic and osteogenic differentiation of BM MSC was demonstrated. In contrast, CB and PBSC mononuclear cells cultured in MSC conditions for two passages produced a population of adherent, non‐confluent fibroblast‐like cells with a haemopoietic phenotype, CD45+, CD14+, CD34–, CD44–, CD90– and CD29–. In paired experiments, cultured BM MSC and mature BM stroma were seeded with CB cells enriched for CD34+. Similar numbers of colony‐forming units of granulocytes–macrophages were produced by MSC‐based and standard stroma cultures over 10 weeks. We conclude that adult BM is a reliable source of functional cultured MSC, but CB and PBSC are not.

Chromosome translocations and covert leukemic clones are generated during normal fetal development

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins (≈5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase–PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10−4 to 10−3) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.

Malignant Tumors Occurring after Treatment of Aplastic Anemia

BACKGROUND AND METHODS Recent studies have shown that long-term survivors of acquired aplastic anemia may be at high risk for malignant diseases. We assessed the risk of cancer after aplastic anemia was treated with immunosuppression or bone marrow transplantation and sought to identify risk factors according to treatment. The study population consisted of 860 patients treated by immunosuppression and 748 patients who had received bone marrow transplants for the treatment of severe aplastic anemia. The risk of cancer was analyzed overall and according to treatment relative to the risk in the general population. In calculating relative risk, we excluded patients with myelodysplastic syndromes or acute leukemias arising less than 6 months after treatment, and solid cancers arising less than 12 months after treatment, because of a possible association with aplastic anemia itself rather than with the treatment received. RESULTS Forty-two malignant conditions were reported in the 860 patients who received immunosuppressive therapy: 19 cases of myelodysplastic syndrome, 15 cases of acute leukemia, 1 case of non-Hodgkins lymphoma, and 7 solid tumors. Nine were reported in the 748 patients who received bone marrow transplants: two cases of acute leukemia and seven solid tumors. After the exclusions listed above, the overall relative risk of cancer was 5.50 (P < 0.001) as compared with that in the general European population; the risk was 5.15 (P < 0.001) after immunosuppressive therapy and 6.67 (P < 0.001) after transplantation. The 10-year cumulative incidence rate of cancer was 18.8 percent after immunosuppressive therapy and 3.1 percent after transplantation. The risk factors for myelodysplastic syndrome or acute leukemia after immunosuppressive therapy included the addition of androgens to the immunosuppressive treatment (relative risk = 0.28), older age (relative risk = 1.03), treatment in 1982 or later, as compared with 1981 or earlier (relative risk = 3.01), splenectomy (relative risk = 3.65), and treatment with multiple courses of immunosuppression (relative risk = 2.26). Risk factors for solid tumors after bone marrow transplantation were age (relative risk = 1.11 per year) and the use of radiation as a conditioning regimen before transplantation (relative risk = 9.56); such tumors occurred only in male patients. CONCLUSIONS Survivors of aplastic anemia are at high risk for subsequent malignant conditions. Myelodysplastic syndrome and acute leukemia tend to follow immunosuppressive therapy, whereas the incidence of solid tumors is similar after immunosuppression and after bone marrow transplantation.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT* SAA Working Party

This is an analysis of 509 patients with severe aplastic anaemia (SAA) treated in Europe between 1981 and 1986; 218 patients were treated by allogeneic bone marrow transplantation (BMT) from HLA identical sibling donors and 291 with immunosuppressive therapy (IS) with antilymphocyte globulin (ALG). The overall actuarial survival was 63% after BMT and 61% after IS therapy at 6 years. All patients fulfilled the criteria of SAA; however, most patients with a neutrophil count of <0.2 × 109/l also had infections and haemorrhages. Therefore a further subclassification was defined by pretreatment peripheral blood neutrophil count: very severe aplastic anaemia (vSAA) (<0.2 × 109/l neutrophils) and moderately severe aplastic anaemia (mSAA) (0.2‐0.5 × 109/l neutrophils).

Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin

Summary. 468 severe aplastic anaemia (SAA) patients registered in the EBMT‐SAA registry who did not undergo bone marrow transplantation and were treated with immunosuppressive therapy (IS; 96% of patients received ATG) were evaluated. Their median age was 23 years (range 1–73) at initial IS therapy, 59% were males; in 69% the aetiology of SAA was idiopathic. Of these 468 patients, 245 had a follow‐up of <2 years after IS 166/245 died, 71/245 are still alive, 8/245 are lost to follow‐up. Of 223 patients who survived ≥2 years (LTS long‐term survivors), 191 are alive, 21 died >2 years and 11 are lost. Median follow‐up of 223 LTS was 4.1 years (range 2.0–10.9). Comparison of 166 patients who died <2 years and 223 LTS revealed no difference at time of initial IS therapy as regards sex, duration of AA, or its aetiology, but the age distribution and, in particular, severity of SAA differed significantly: more LTS were between 21 and 40 years old (44%v. 32%, P<0.02), less LTS had reticulocytes <20 × 109/l (63%v. 80%, P<0.001), polymorphonuclear granulocytes (PMN) <0.2 × 109/l (30%v. 57%. P<0.001), haemorrhages (58%v. 79%, P<0.002) and infection (30%v. 49%, P<0.005) at time of IS. A gradual improvement of blood counts was seen in patients alive ≥ 2 years after IS. At 2 years after IS 80% had a normal haemoglobin and PMN >0.5 × 109/l, but only after 5 years 80% of cases had platelets > 50 × 109/l. Development of clonal disease was reported of 31 LTS: 19 developed paroxysmal nocturnal haemoglobinuria (PNH), one acute leukaemia, 11 myelodysplastic syndromes and of these 11 five subsequently acute leukaemia. The majority of these patients (23/31) are still alive. Actuarial mortality of LTS is 22% at 8 years, but so far no plateau was achieved. It is concluded that SAA patients who become LTS following IS, show an improvement in haematological status but are probably not cured and are prone to develop clonal (malignant) disease.

Endothelial cell precursors are normal components of human umbilical cord blood

Endothelial cells are part of the normal bone marrow stroma. We have previously shown human umbilical cord blood (UCB) does not produce stroma in standard long‐term cultures. Highly enriched (93–98%) UCB CD34+ cells were cultured for 6 weeks with interleukin‐2 and conditioned medium from the 5637 carcinoma cell line (n = 4). The resulting ‘fibroblast like’ cells were shown to be endothelial by expression of von Willebrand factor (VWF), ICAM‐1 (CD54), E‐selectin (CD62E) and PECAM (CD31). Endothelial monolayers seeded with CD34+ UCB cells supported expansion of colony forming cells and CD34+ cells. We conclude that endothelial cell precursors circulate in UCB, and may be derived from the CD34+ cell fraction.

Bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (MSCs) contribute to the regeneration of mesenchymal tissues, and are essential in providing support for the growth and differentiation of primitive hemopoietic cells within the bone marrow microenvironment. Techniques are now available to isolate human MSCs and manipulate their expansion in vitro under defined culture conditions without change of phenotype or loss of function. Mesenchymal stem cells have generated a great deal of interest in many clinical settings, including that of regenerative medicine, immune modulation and tissue engineering. Studies have already demonstrated the feasibility of transplanted MSCs providing crucial new cellular therapy. In this review, many aspects of the MSC will be discussed, with the main focus being on clinical studies that describe the potential of MSCs to treat patients with hematological malignancies who are undergoing chemotherapy and/or radiotherapy.

Matched-related donor transplantation for sickle cell disease: report from the Center for International Blood and Transplant Research

We report outcomes after myeloablative haematopoietic cell transplantation (HCT) from human leucocyte antigen (HLA)‐matched sibling donors in 67 patients with sickle cell disease transplanted between 1989 and 2002. The most common indications for transplantation were stroke and recurrent vaso‐occlusive crisis in 38% and 37% of patients respectively. The median age at transplantation was 10 years and 67% of patients had received >10 red blood cell transfusions before HCT. Twenty‐seven percent of patients had a poor performance score at transplantation. Ninety‐four percent received busulfan and cyclophosphamide‐containing conditioning regimens and bone marrow was the predominant source of donor cells. Most patients achieved haematopoietic recovery and no deaths occurred during the early post‐transplant period. Rates of acute and chronic graft‐versus‐host disease were 10% and 22% respectively. Sixty‐four of 67 patients are alive with 5‐year probabilities of disease‐free and overall survival of 85% and 97% respectively. Nine patients had graft failure with recovery of sickle erythropoiesis, eight of who had recurrent sickle‐related events. This report confirms and extends earlier reports that HCT from HLA‐matched related donors offers a very high survival rate, with few transplant‐related complications and the elimination of sickle‐related complications in the majority of patients who undergo this therapy.

Relapse of aplastic anaemia after immunosuppressive treatment: a report from the European Bone Marrow Transplantation Group SAA Working Party

Summary This study was designed to determine the incidence of relapse and factors predictive for relapse in 719 patients with severe aplastic anaemia (SAA) after immunosuppressive treatment (IS). Patients developing myelodysplasia or acute leukaemia after IS, and patients receiving a transplant, were excluded from this analysis. Response was defined as reaching complete independence from transfusions, relapse was defined as becoming again transfusion dependent. This criteria was validated by similar figures when using other ‘relapse criteria’ such as drop in neutrophil or platelet counts.