Quantitative RT-PCR analysis of the MOZ-CBP fusion transcript in therapy-related acute myeloid leukemia with t(8;16)(p11;p13).

We developed a real time reverse transcriptase polymerase chain reaction (RT-PCR) assay system for detecting the MOZ-CBP fusion transcript and used it to monitor minimal residual disease (MRD) status in a patient with therapy related acute myeloid leukemia (t-AML) harboring t(8;16)(p11;p13). Expression of the MOZ-CBP fusion transcript was determined by RT-PCR analysis of the patient's bone marrow at the time of diagnosis. Thereafter, real time RT-PCR was used to evaluate MRD levels throughout the entire course of treatment. The sensitivity of quantitative RT-PCR for the MOZ-CBP fusion transcript was 10(-5). Below this level, MRD was classified as negative. Real time RT-PCR of the bone marrow after induction therapy showed the reduction of MOZ-CBP transcript to approximately 10(-3) level when compared to the diagnostic sample. MRD was classified as negative (< 10(-5) compared with that in the bone marrow at diagnosis) after 5 courses of chemotherapy, a level that was maintained post-allo-hematopoietic stem cell transplantation. Real time RT-PCR of the MOZ-CBP transcript is a useful tool for assessing MRD status for a patient with therapy related acute myeloid leukemia who was initially predicted to have a poor prognosis.

[Effective infliximab treatment for a recurrent type of acute intestinal graft-versus-host disease accompanied by steroid-induced depression].

A 22-year-old man with chronic active Epstein-Barr virus infection underwent allogeneic bone marrow transplantation (allo-BMT) from an HLA two allele-mismatched unrelated donor. Ten months after allo-BMT, he developed protein-losing enteropathy following a respiratory syncytial virus infection. A diagnosis of a recurrent type of acute graft-versus-host disease (GVHD) was made based on the histopathological findings, such as the infiltration of T lymphocytes into the superficial epithelium and crypts, and apoptotic bodies in crypts. Although methylprednisolone (mPSL: 10 mg/kg) administration for two consecutive days improved gastrointestinal symptoms, acute pancreatitis and severe depression developed in association with corticosteroid treatment. Reduction of mPSL and administration of infliximab (5 mg/kg/dose, 3 times) resulted in rapid improvement of depression and pancreatitis without aggravating intestinal GVHD. Recent studies have demonstrated that tumor-necrosis-factor (TNF)-α is associated with not only GVHD but also depression and acute pancreatitis. In the present case, anti-TNF-α treatment enabled us to reduce corticosteroid dose without aggravating GVHD, which suggests that this approach might be effective for the treatment of depression and acute pancreatitis.

Evaluation of angiopoietins and cell-derived microparticles after stem cell transplantation.

Although stem cell transplantation (SCT) is being used for hematopoietic reconstitution following high-dose chemotherapy for malignancy, it involves certain serious transplant-related complications such as graft-versus-host disease (GVHD). Angiopoietins play important roles in angiogenesis. However, the role of angiopoietins after SCT is poorly understood. In this study, 52 patients underwent SCT; 26 patients received allogeneic SCT, while the remaining 26 received autologous SCT. In 48 of 52 patients, levels of angiopoietins, cytokines, and soluble factors were measured by enzyme-linked immunosorbent assay. Soluble Fas ligand (sFasL) and endothelial cell-derived microparticle (EDMP) exhibited significant elevation in the early phase (2-3 weeks) after SCT. In addition, the elevation of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and sIL-2 receptor (sIL-2R), which are GVHD markers after allogeneic SCT was observed. The level of angiopoietin (Ang)-2 in allogeneic SCT continued to increase for up to 4 weeks, although the level of Ang-1 did not show significant changes. The patients with high Ang-2 exhibited significant increase of sFasL and EDMP compared with those with low Ang-2. In addition, the patients with high-grade GVHD exhibited a significant increase in Ang-2 compared to patients with low-grade GVHD. In the in vitro experiment using endothelial cells, the suppressive effect of Ang-1 on EDMP generation by TNF-alpha was partially inhibited by the addition of Ang-2. Furthermore, multivariate regression analysis showed that EDMP and sFasL were significant factors in Ang-2 elevation. Our results suggest that Ang-2 generation after allogeneic SCT relates to GVHD.

Role of soluble tumor necrosis factor-related apoptosis-inducing ligand concentrations after stem cell transplantation.

Although stem cell transplantation (SCT) is being used for hematopoietic reconstitution following high-dose chemotherapy for malignancy, it involves certain serious transplant-related complications such as graft-versus-host disease (GVHD). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays important roles in regulating cell death, immune response, and inflammation. However, the role of soluble TRAIL (sTRAIL) after SCT is poorly understood. In this study, 42 patients underwent SCT; 22 patients received allogeneic SCT, while the remaining 20 received autologous SCT. In these patients, levels of sTRAIL, cytokines, and soluble factors were measured by enzyme-linked immunosorbent assay (ELISA). In addition, a basic study of the generation of endothelial cell-derived microparticle (EDMP) by TNF-alpha and soluble Fas ligand (sFasL) was conducted. sFasL and EDMP exhibited significant elevation in the early phase (2-3 weeks) after SCT. In addition, the elevation of IL-6, TNF-alpha, and sIL-2R after allogeneic SCT was observed. EDMP also exhibited changes similar to sFasL. The patients with high sTRAIL exhibited significant decrease of sFasL and EDMP as compared with those without high sTRAIL. TNF-alpha and sFasL induced an increase in procoagulant and apoptotic markers in endothelial cells, and EDMP shedding was observed. Furthermore, sTRAIL inhibited the EDMP elevation caused by TNF-alpha and sFasL. The apoptotic markers such as sFasL and sTRAIL exhibited particular changes after SCT. Our results suggest that sTRAIL generation after allogeneic SCT relates to the prevention of GVHD.

Outcome of non-T-cell-depleted HLA-haploidentical hematopoietic stem cell transplantation from family donors in children and adolescents.

Non-T-cell-depleted HLA-haploidentical hematopoietic stem cell transplantation (SCT) from family members has been reported, but its effectiveness and safety are not fully known. In this study, we examined the outcomes of 83 children and adolescents with nonmalignant (n = 11) or malignant (n = 72) disorders who underwent SCT mismatched at 2 or 3 HLA loci, either from the mother (n = 56), a noninherited maternal antigen (NIMA)-mismatched sibling (n = 14), or the father/a noninherited paternal antigen (NIPA)-mismatched sibling (n = 13). Engraftment was satisfactory. Severe (grade III-IV) acute graft-versushost disease (GVHD) was noted only in malignant disease, with an incidence of 21 of 64 evaluable patients. GVHD prophylaxis with a combination of tacrolimus and methotrexate was significantly associated with a lower risk of severe acute GVHD, compared with other types of prophylaxis (P = .04). Nine of 11 patients with nonmalignant disease and 29 of 72 patients with malignant disease were alive at a median follow-up of 26 months (range, 4-57 months). Outcomes were not significantly different among the 3 donor groups (mother versus NIMA-mismatched sibling versus father/NIPA-mismatched sibling) for the malignancy disorders. Our results indicate that non-T-cell-depleted HLA-haploidentical SCT may be feasible, with appropriate GVHD prophylaxis, for young recipients who lack immediate access to a conventional stem cell source.

Role of platelet-derived chemokines (RANTES and ENA-78) after stem cell transplantation.

Stem cell transplantation (SCT) is being used for hematopoietic reconstitution following high-dose chemotherapy for malignancy. Some patients seem to have an imbalance of the immune response after SCT and cytokines are known to regulate this response. Recently, platelets have been shown to contain members of the chemokine family, suggesting a role of platelets as inflammatory cells. We measured and compared levels of platelet activation markers, chemokines, and soluble factors in patients undergoing SCT. IL-8 and GROalpha exhibited a significant elevation in the early phase (1 or 2 weeks) after SCT; this trend was marked after autologous SCT. Furthermore, these levels significantly and positively correlated with the change in G-CSF. In contrast, ENA-78 exhibited a significant elevation in the later phase (3 or 4 weeks) after SCT. In addition, its level negatively correlated with the change in G-CSF. Soluble CD40 ligand and platelet-derived microparticles significantly increased after both auto- and allo-SCT. In addition, ENA-78 positively correlated with the level of platelet-derived microparticles. The increase of RANTES seems to be related to platelet activation, since RANTES was in the dynamic phase similar to soluble CD40 ligand and platelet-derived microparticles. RANTES exhibited changes similar to IL-6, TNFalpha, and soluble IL-2 receptors, which are GVHD markers. Thus, the platelet-derived chemokines ENA-78 and RANTES exhibited particular changes after SCT. Our results suggest that ENA-78 play a role in hematopoietic conditions in which G-CSF is not involved, and RANTES generation after allo-SCT relates to GVHD.

Successful autologous peripheral blood stem cell transplantation with rituximab administration for pediatric diffuse large B-cell lymphoma.

A 13-year-old boy with chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) was successfully treated with autologous peripheral blood stem cell transplantation (auto-PBSCT) with administration of rituximab. Previous reports indicate that auto-PBSCT without rituximab for adult chemotherapy-resistant DLBCL is only marginally successful. The addition of rituximab administration might have intensified anti-tumor activity before the transplant procedure and might have enhanced the in vivo purging of the auto-graft, resulting in a successful outcome in this case. Although a few adverse effects are linked to rituximab administration, such as prolonged neutropenia, hypogammaglobulinemia, and increased infectious complications, the regimen of rituximab with SCT appears to be effective against chemotherapy-resistant DLBCL.

Prognostic value of early response to treatment combined with conventional risk factors in pediatric acute lymphoblastic leukemia.

To determine useful prognostic factors in treating childhood acute lymphoblastic leukemia (ALL), we correlated conventional risk factors and bone marrow response 14 days after induction chemotherapy. Our study included 116 precursor B-cell (n = 104) and T-cell (n = 12) ALL patients treated with our protocol between 1988 and 1999. The patients were classified into 3 initial risk groups on the basis of conventional risk factors (56 in the low-risk, 33 in the high-risk, and 27 in the very high-risk groups). All patients received similar systemic chemotherapy regimens before the evaluation of their bone marrow on day 14. We evaluated the marrow of 69 patients as M1 (less than 5% blasts), 25 as M2 (5%-25% blasts), and 22 as M3 (more than 25% blasts). Although all patients attained an initial complete remission (CR), relapse was noted in 33 of the 116 patients, and 15 patients died. All of the M1 marrow patients, irrespective of the initial risk group, showed the best event-free survival rate (85.1% +/- 3 4.4%), the lowest relapse rate (14.5%), and the highest attainment of a second CR (100%); they were defined as the new R1 prognostic group. The low-risk patients with M2 or M3 marrow (R2 group) had a relatively high relapse rate, but all of these relapsed patients were treated successfully with subsequent therapy. High- or very high-risk patients with M2 or M3 marrow (R3 group) had the worst prognosis. Our new prognostic definition (R1, R2, R3) incorporating day 14 marrow findings is useful to tailor early-phase treatments for better therapeutic results in childhood ALL.

[A woman complicated with immune thrombocytopenic purpura, subclinical Graves disease and peripheral neuropathy 5 years after allogeneic bone marrow transplantation].

A 21-year-old woman developed immune thrombocytopenia (ITP), subclinical Graves disease and peripheral neuropathy without typical chronic graft-versus-host disease (GVHD) 5 years following an allogeneic bone marrow transplantation from an HLA-identical sibling. She received high-dose intravenous immunoglobulin (IVIG) and prednisolone (PSL), which resulted in transient recovery of platelet numbers and muscle weakness. A combination of cyclosporine and PSL induced a durable response against not only the thrombocytopenia but also her high levels of thyroid stimulating antibody (TSAb), muscle weakness and sensory abnormality. The level of thyroglobulin in the donor, who had not developed Graves disease, was also elevated, indicating that late onset-subclinical Graves disease was caused by donor lymphocytes that were autoreactive to the thyroid glands.

Sister and brother with Vici syndrome: agenesis of the corpus callosum, albinism, and recurrent infections.

A sister and brother with Vici syndrome are described. They both had oculocutaneous albinism, agenesis of the corpus callosum, cataracts, and cardiomyopathy. They were born to healthy unrelated parents, and had postnatal growth retardation, profound developmental delay, hypotonia, and cataracts. The sister had recurrent infections, and died of progressive heart failure at age 19 months. The brother is alive at age six months with mild cardiomyopathy, and had a single episode of acute bronchitis at age three months. Review of the clinical manifestations of the sibs we described and six children reported in the literature indicates that Vici syndrome is a distinct clinical entity. Its main clinical manifestations include growth retardation, profound developmental delay, hypotonia, albinism, agenesis of the corpus callosum, cataracts, cardiomyopathy, and recurrent infections. The occurrence of the syndrome in three pairs of sibs of both sexes born to unaffected parents supports autosomal recessive inheritance.

Granulocytic sarcoma presenting with severe adenopathy (cervical lymph nodes, tonsils, and adenoids) in a child with juvenile myelomonocytic leukemia and successful treatment with allogeneic bone marrow transplantation.

The occurrence of adenopathy in patients with myelodysplastic syndrome-associated extramedullary myeloid cell tumors has rarely been reported. We describe a 7-year-old girl with juvenile myelomonocytic leukemia who showed the novel chromosomal abnormality t(9;12)(p22;q24.1) and who developed severe adenopathy of the cervical lymph nodes, tonsils, and adenoids that was manifested as granulocytic sarcoma. Following chemotherapy, the patient underwent a conditioning regimen of busulfan, cyclophosphamide, and total body irradiation followed by successful allogeneic bone marrow transplantation from her single HLA locus-mismatched mother at 6 months after her diagnosis. The patient continues to be well and in remission 3 years after stem cell transplantation.

A preparatory regimen of total body irradiation, busulphan and melphalan for allogeneic bone marrow transplantation in childhood high-risk leukemia and lymphoma.

The therapeutic results of allogeneic bone marrow transplantation (BMT), following a conditioning regimen of total body irradiation and busulphan and melphalan administration, were evaluated in 20 pediatric patients with high-risk leukemia or lymphoma. Twelve patients received BMT from HLA-matched related (MR) donors while eight received transplants from mismatched related or unrelated (MisR/UR) donors. The post-BMT five-year survival rates were much better for patients in the MR donor group (p = 0.0008). The outcomes of patients in the MisR/UR donor group were significantly worse. This was not due to disease recurrence, but to a high incidence of fatal post-transplant infections (p = 0.004). Nine out of twelve patients who received transplants from MR donors have remained in complete remission for a median of 57 (range 27-78) months. These results suggest that this conditioning regimen has a significant anti-neoplastic benefit useful for the preparation of pediatric patients receiving transplants from MR donors; however, refinement is essential before it can be used in patients receiving transplants from MisR/UR donors.

[Acute lymphoblastic leukemia in childhood treated with non-T-cell depleted bone marrow transplantation from an HLA 2 loci (HLA-A and-DRB1)-mismatched mother after early graft failure of cord blood transplantation].

An 11-year-old boy with acute lymphoblastic leukemia received unrelated cord blood transplantation at the second remission. Because of early graft failure, he was given a second non-T-cell depleted bone marrow transplant from his HLA 2 loci (HLA-A and -DRB1)-mismatched mother 36 days after the first transplantation. Feto-maternal microchimerism was verified before transplantation. The second transplantation was performed with fludarabine/melphalan as a conditioning regimen, and tacrolimus/short-course methotrexate as graft-versus-host disease (GVHD) prophylaxis. Engraftment was prompt with a recovery of neutrophils (> 0.5 x 10(9)/1) by day +10, reticulocytes (> 1%) by day +17 and platelets (> 50 x 10(9)/l) by day +18. Mild regimen-related toxicities (grade I gastrointestinal, grade II hepatic) were observed and acute GVHD was grade I (skin: stage 2). No severe complication was noted. At 6 months post-transplantation, he had no chronic GVHD or leukemia relapse. This experience indicates the future feasibility of a back-up non-T-cell depleted transplantation from HLA 2 loci-mismatched and feto-maternal microchimerism-positive mothers in cases with primary graft failure.

[Granulocyte transfusions for severe infections prior to allogeneic hematopoietic stem cell transplantation].

The efficacy and safety of granulocyte transfusions were evaluated in two acute lymphoblastic leukemia patients for the control of severe infections (cervical cellulitis, sepsis) prior to hematopoietic stem cell transplantation. One patient received 6 transfusions and the other 2 transfusions. The donors were given subcutaneous granulocyte-colony stimulating factor plus oral dexamethasone/betamethasone 12 hours before the scheduled collection. Granulocytes were obtained by standard leukapheresis procedures utilizing hydroxyethyl starch with processing of 7 liters of blood. The yield was 3.2-10.7 x 10(10) (0.7-2.1 x 10(9)/kg of recipient) granulocytes. Post-transfusion increases of peripheral blood neutrophil counts in the following morning were 300 to approximately 6,900/ml. Infections resolved and successful engraftment was obtained in both patients after the transplants. No severe adverse reactions were observed. These findings suggest that granulocyte transfusions are useful for control of severe infections prior to allogeneic hematopoietic stem cell transplantation.

Complications of Evans' syndrome in an infant with hereditary spherocytosis: a case report.

Hereditary spherocytosis (HS) is a genetic disorder of the red blood cell membrane clinically characterized by anemia, jaundice and splenomegaly. Evans' syndrome is a clinical syndrome characterized by autoimmune hemolytic anemia (AIHA) accompanied by immune thrombocytopenic purpura (ITP). It results from a malfunction of the immune system that produces multiple autoantibodies targeting at least red blood cells and platelets. HS and Evans' syndrome have different mechanisms of pathophysiology one another. We reported the quite rare case of an infant who had these diseases concurrently. Possible explanations of the unexpected complication are discussed.

A randomized prospective study on the use of 2 g-IVIG or 1 g-IVIG as therapy for Kawasaki disease.

A single, 2 g/kg dose of immune globulin (IG), denoted 2 g-intravenous (IV)IG, has become a standard regimen for treating Kawasaki disease (KD) because of its highly preventive effect on coronary arterial lesions (CAL). However, IG is obtained from blood specimens, a drawback to many patients, and is also very expensive. This randomized prospective study reported here was carried out with the aim of developing a treatment regimen that would reduce the total dose of IG. The study tested two protocols (A: 2 g-IVIG; B: 1 g-IVIG) that included the strategy of administering additional IVIG to IVIG-resistant patients based on the criteria we described previously. In protocol A, an additional 2 g-IVIG was administered only once; in protocol B, the first additional IVIG was 1 g-IVIG and the second was 2 g-IVIG. One hundred and nine patients who were admitted before the seventh day of illness and had no CAL at the time of admission were enrolled in the study (protocol A: 54 patients; B: 55 patients). In the protocol A group, 7.4% (4/54) of the patients received 4 g/kg IG. In protocol B, 41.8% (23/55) were treated only with 1 g/kg IG, and 10.9% (6/55) received 4 g/kg IG. No significant differences were observed between the patients of the two subgroups receiving 4 g/kg IG in each protocol group. Discriminate analysis also suggested that 52.4% of the patients in the protocol A group could be treated only with 1 g/kg IG. On the other hand, no significant difference was observed in the incidence of aneurysms between patients in the protocol A group (1/54) and those in the protocol B group (4/55). Our protocol based on 1 g-IVIG, including additional IVIG, was assessed to be an effective treatment and to provide a considerably useful means to reduce the total dose of IG.

First description of somatic mosaicism in MYH9 disorders.

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Dohle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, which encodes non-muscle myosin heavy chain-A (NMMHCA). These disorders are known to be transmitted in an autosomal dominant manner, although about 20% of cases are considered to be sporadic. We report here the first case of a MYH9 disorder because of somatic mosaicism. The patient was the father of a male with typical May-Hegglin anomaly. The father had normal platelet counts, however, both normal-sized and giant platelets were observed on his peripheral blood smears. In addition, 14% of neutrophils contained inclusion bodies and the rest showed a normal morphology. Quantitative fluorescent polymerase chain reaction analysis showed that only 6% of DNA from peripheral blood leucocytes harboured the mutation. The mosaicism was demonstrated at a similar rate in different tissues, buccal mucosa cells and hair bulb cells, implying that the mutation had occurred before gastrulation. Mosaicism might account for some de novo mutations in MYH9 disorders.

Invasive adenoviral infection in a recipient of unrelated bone marrow transplantation: Problems with diagnostic PCR.

A hematopoietic stem cell transplantation (SCT) recipient developed severe diarrhea and fever. A rapid test for the presence of adenovirus (AdV)-specific antigen in the patient's stools was positive; however, AdV genome was not detected by conventional or real-time polymerase chain reaction (PCR). AdV was confirmed by specific PCR for AdV serotype 7 and by an AdV hexon/fiber gene DNA sequence homology search of the PCR product. We suspect that conventional/real-time PCR failed to detect AdV due to nine silent single base substitutions in the extracted AdV genome. Treatment with 1 mg/kg cidofovir (CDV) intravenously three times a week was effective.

Successful transplantation of haploidentical CD34+ selected bone marrow cells for an infantile case of severe combined immunodeficiency with aspergillus pneumonia.

A 5-month-old boy with X-linked severe combined immunodeficiency (SCID) and aspergillus pneumonia was successfully transplanted. Before and during transplantation, the patient received O2 administration, systemic amphotericin B, and itraconazole. The transplant was performed with a conditioning regimen of busulfan/cyclophosphamide and 2.9 x 10(6)/kg of CD34+ selected bone marrow cells from his HLA haploidentical mother. Acute grade II graft-versus-host disease (GvHD) was well controlled. Neutrophil counts reached >0.5 x 10(9)/L by day 15 and platelet counts reached > 50 x 10(9)/L by day 48. The T-cell subset (counts) in peripheral blood increased to 42.2% (0.31 x 10(9)/L) by day 46. The pneumonia improved by day 54. The patient has been doing well with limited chronic GvHD of the gut with a follow-up of longer than 40 months after BMT. Conquest of aspergillus pneumonia in SCID infants could be achieved by CD34+ bone marrow cell transplantation together with appropriate anti-fungal treatment.