Oxidative stress levels in myelodysplastic syndrome patients: their relationship to serum ferritin and haemoglobin values.

Reactive oxygen species (ROS) and serum ferritin levels are both considered to be important biological factors in the pathogenesis of myelodysplastic syndrome (MDS). This study evaluated the levels of ROS in 40 patients with MDS (19 males and 21 females) using the Free Radical Analytical System, FRAS4, and derivatives of reactive oxygen metabolite kits. The patients' mean age was 67.3 years (range 58 - 86 years). The sera of 34 (85%) patients exhibited higher levels of oxidative stress than the reference range. There was a positive correlation between ROS levels and serum ferritin levels, and a negative correlation between ROS levels and haemoglobin levels. There was a negative relationship between serum haemoglobin and ferritin levels. The results indicated that iron accumulation or severe anaemia could contribute to oxidative stress in MDS patients. Iron chelation and antioxidant therapy may be suitable for the management of MDS.

Allogeneic stem cell transplantation in children with acute lymphoblastic leukemia after isolated central nervous system relapse: our experiences and review of the literature.

The prognosis of patients with acute lymphoblastic leukemia (ALL) and central nervous system (CNS) relapse has historically been very poor. Although chemo-radiotherapy has improved outcomes, some patients still have a poor prognosis after CNS relapse. Therefore, allogeneic hematopoietic stem cell transplantation (allo-SCT) has recently become an option for treatment of CNS leukemia; however, information, particularly on the long-term outcome of transplant recipients, is limited. We performed allo-SCT in eight pediatric patients with ALL (n=7) or T-cell type non-Hodgkin's lymphoma (n=1), who had isolated CNS relapse. All patients survived for a median of 70.5 (range, 13-153) months after SCT. Sequelae developed late in some patients: mental retardation (IQ=47) in one patient, severe alopecia in two patients, limited chronic graft-versus-host-disease in three patients, and amenorrhea and/or hypothyroidism in three patients. Except for a pre-school child with post transplant CNS relapse, six out of seven patients show normal school/social performance. Our results clearly indicate a high cure rate of isolated CNS relapse by allo-SCT in pediatric lymphoid malignancies; however, there needs to be further studies to determine which are the appropriate candidates for transplantation and what is the best transplant regimen to achieve high cure rate and maintain good quality of life.

Numerous nonclonal chromosomal aberrations arising in residual recipient hematopoietic cells following allogeneic bone marrow transplantation.

A young female patient in a second remission of acute lymphoblastic leukemia underwent bone marrow transplantation after total body irradiation and high-dose cytarabine from her HLA-matched brother. Following successful engraftment, mixed chimerism was seen 75 days post transplant. The karyotype contained numerous abnormalities in residual recipient cells. Chromosomes 1, 7, 13, and X were significantly more affected than other chromosomes. The high-frequency breakpoints identified were 1p22.2, 5q31.2, and 13q14.2. Some karyotypes specific for leukemia, such as t(9;22)(q34.1;q11.2) and t(8;21)(q22.2;q22.2), not seen with the original disease, were also present. As the frequency of aberrant chromosomes increased markedly with time, donor leukocytes were infused 14 months after BMT, which effectively eradicated the abnormal karyotypes.

High frequency of QPY allele and linkage disequilibrium of granzyme-B in Epstein-Barr-virus-associated hemophagocytic lymphohistiocytosis.

Mediation of Epstein-Barr virus (EBV)-specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme-B (55Q/R, 95P/A and 247Y/H) in patients with EBV-HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV-HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme-B might be responsible for the pathogenesis of EBV-HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme-B but also by other molecules, including other granzymes or Fas.

Identification of novel MUNC13-4 mutations in familial haemophagocytic lymphohistiocytosis and functional analysis of MUNC13-4-deficient cytotoxic T lymphocytes.

BACKGROUND: Familial haemophagocytic lymphohistiocytosis (FHL) has an autosomal recessive mode of inheritance and consists of at least three subtypes. FHL2 subtype with perforin (PRF1) mutation accounts for 30% of all FHL cases, while FHL with MUNC13-4 mutation was recently identified and designated as FHL3 subtype. OBJECTIVE: To examine MUNC13-4 mutations and the cytotoxic function of MUNC13-4 deficient T lymphocytes in Japanese FHL patients METHODS: Mutations of MUNC13-4 and the cytotoxicity of MUNC13-4-deficient cytotoxic T lymphocytes (CTL) were analysed in 16 Japanese families with non-FHL2 subtype. RESULTS: Five new mutations of the MUNC13-4 gene were identified in six families. The mutations were in the introns 4, 9, and 18, and exons 8 and 19. Two families had homozygous mutations, while the remaining four had compound heterozygous mutations. Cytotoxicity of MUNC13-4 deficient CTL was low compared with control CTL, but was still present. Clinically, the onset of disease tended to occur late; moreover, natural killer cell activity was not deficient in some FHL3 patients. CONCLUSIONS: MUNC13-4 mutations play a role in the development of FHL3 through a defective cytotoxic pathway.

Adenoviral infection in hematopoietic stem cell transplantation: early diagnosis with quantitative detection of the viral genome in serum and urine.

Early diagnosis and prompt introduction of effective therapy are imperative to manage systemic, often fatal adenoviral (AdV) disease following hematopoietic stem cell transplantation (SCT). We evaluated the usefulness of real-time polymerase chain reaction (PCR) in the diagnosis of AdV disease in SCT recipients. Seven SCT recipients, including three with AdV disease, were retrospectively evaluated for AdV genome detection. In serum specimens, the AdV genome was detected at >10(3) copies/ml in the pre-SCT period in two of the five recipients studied. These two patients subsequently developed AdV disease. The three patients with AdV disease had high levels of >10(5) copies/ml during the 4-6 weeks post-SCT period. In none of these patients was the AdV genome detected in urine specimens in pre-SCT period. However, three recipients with detectable urinary levels during the period 1-2 weeks post-SCT subsequently developed AdV disease. Regarding the outcome, two of the three patients with AdV disease died of progressive renal failure. Our results suggest that quantitative determination of the AdV genome in serum and urine is useful to identify patients at high risk of developing AdV disease. Prospectively applied, these measures are expected to improve the dismal outcome of AdV disease in SCT recipients.

Lack of clinical utility of minimal residual disease detection in allogeneic stem cell recipients with childhood acute lymphoblastic leukemia: multi-institutional collaborative study in Japan.

The clinical utility of minimal residual disease (MRD) measurements following allogeneic stem cell transplantation (SCT) in childhood ALL is controversial. We therefore performed a multi-institutional study of MRD in bone marrow samples taken before SCT and at 1, 3, 6 and 12 months after SCT. Case-specific clonal rearrangements of IgH and TCR genes and expression levels of Wilms' tumor 1 (WT1) mRNA were determined by PCR or RT-PCR methods. In total, 95 cases met all criteria for analysis of informative IgH/TCR markers and quantitative WT1 mRNA expression levels. During the 2-year (median 414 days) study period, 20 patients relapsed. Although the proportion of patients with a positive IgH/TCR result before SCT was significantly reduced at 1 month after treatment (P<0.001), attesting the efficacy of SCT, serial measurements of IgH/TCR rearrangements did not correlate with leukemic relapse. Clonal switch was demonstrated in 11 of the 14 patients with bone marrow relapse, indicating that the poor predictive power of the MRD assay most likely reflected the loss of PCR targets. WT1 expression was not related to either MRD detection by IgH/TCR assays or to clinical leukemic relapse. The clinical value of serial MRD monitoring would be limited in ALL patients undergoing SCT.

Acute renal failure due to adenovirus-associated obstructive uropathy and necrotizing tubulointerstitial nephritis in a bone marrow transplant recipient.

Management of post-transplant complications caused by severe adenoviral infection remains a major therapeutic challenge. A 17-year-old male who had undergone bone marrow transplantation for the treatment of acute lymphoblastic leukemia developed complete anuria following hemorrhagic cystitis 34 days after the transplant procedure. The computed tomogram scan revealed bilateral hydronephrosis, indicating acute renal failure because of obstructive uropathy. The emergency procedure of percutaneous nephrostomy caused massive bleeding in the left kidney, which eventually required a nephrectomy. Adenovirus-positive severe necrotizing tubulointerstitial nephritis was the histopathological diagnosis. Post-transplant acute renal failure because of hydronephrosis, which could be complicated by adenovirus-induced renal parenchymal disease, is of great concern and may cause significant problems with interventional treatment.

A novel infant acute lymphoblastic leukemia cell line with MLL-AF5q31 fusion transcript.

Infant acute lymphoblastic leukemia (ALL) is characterized by the presence of the proB phenotype (CD10(-)/CD19(+)), poor prognosis and frequent rearrangement of the mixed lineage leukemia (MLL) gene. The most frequent rearrangement is t(4;11)(q21;q23), the role of whose product, the MLL-AF4 fusion transcript, has been extensively studied in leukemogenesis. In a cell line of infant leukemia with MLL rearrangement denoted KP-L-RY, panhandle PCR amplification of cDNA revealed the presence of a fusion transcript, MLL-AF5q31, indicating that AF5q31 is also a partner gene of MLL. In this fusion transcript the MLL exon 6 is fused in frame to the 5' side of the putative transactivation domain of AF5q31. The AF5q31 protein is a member of the AF4/LAF4/FMR2-related family of proteins, which have been suggested to play a role in hematopoietic cell growth and differentiation. The MLL-AF5q31 fusion transcript, although probably rare, appears to be associated with the pathogenesis of infant ALL like MLL-AF4. Co-expression of HoxA9 and Meis1 genes in the KP-L-RY cell line indicated possible functional similarity between MLL-AF4 and MLL-AF5q31. Further understanding of the function of AF5q31 as well as the specific leukemogenic mechanism of MLL-AF5q31 awaits future studies.

Epstein-Barr virus-associated T-lymphoproliferative disease with hemophagocytic syndrome, followed by fatal intestinal B lymphoma in a young adult female with WHIM syndrome. Warts, hypogammaglobulinemia, infections, and myelokathexis.

A rare association of Epstein-Barr virus-associated T- and B-lymphoproliferative disease (EBV(+) T- and EBV(+) B-LPD) in a patient with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is reported. A 26-year-old Japanese female, who had been treated for WHIM syndrome since early childhood, developed hemophagocytic syndrome associated with EBV(+) T-LPD at the lymph nodes and spleen. The disease rapidly resolved in response to prednisolone therapy. However, 6 weeks later, fatal EBV(+) B lymphoma unresponsive to chemotherapy occurred in the intestine and other organs. Caution must be exercised that the patient with WHIM syndrome may be at risk for EBV-LPD.

Recent developments in the management of haemophagocytic lymphohistiocytosis.

Over the past two decades, the underlying pathophysiology of haemophagocytic lymphohistiocytosis (HLH) (synonyms: haemophagocytic syndrome, macrophage activation syndrome) has been well recognised. Cytokine storm plays a major role, which derives from an inappropriate immune reaction caused by proliferating and activated T-cell or natural killer (NK) cells associated with macrophage activation and inadequate apoptosis of immunogenic cells. Many biological parameters reflecting activity of disease or response to treatment have been identified, in particular, serum ferritin has been confirmed to be one of the markers for HLH. The common types of HLH consist of non-hereditary (acquired) infection-associated disease such as Epstein-Barr virus (EBV)-haemophagocytic lymphohistiocytosis (HLH) and hereditary (familial) disease such as FHL, in which, at the molecular level, dysfunctional perforin was clarified. Regarding the therapeutic strategies, prompt differential diagnosis of underlying disease is essential and choice of treatment should be based on the risk (low or high) of prognosis, where either cyclosporin A, steroids or iv. immunoglobulin (IVIG) may be indicated as initial treatment for low-risk patients, with etoposide-containing regimens for high-risk patients. Significant improvement of prognosis has been obtained by incorporating intensive supportive care at the disease onset and prompt introduction of immunosuppressants to control cytokine storm. Subsequent immunochemotherapy and haemopoietic stem cell transplantation have contributed significantly to further improve survival of hereditary and refractory HLH patients.

[AML(M7) associated with t(16;21)(p11;q22) showing relapse after unrelated bone marrow transplantation and disappearance of TLS/FUS-ERG mRNA].

A 3-year-old boy with poorly prognostic acute megakaryoblastic leukemia (AML M7) showing t(16;21)(p11;q22) karyotype underwent unrelated bone marrow transplantation (U-BMT) during his first hematological remission. The conditioning regimen consisted of BU, VP-16 and L-PAM. Engraftment was smooth, but the patient developed grade I acute GVHD. During hematological remission before U-BMT, the TLS/FUS-ERG chimeric transcript of t(16;21)(p11;q22) was consistently detectable as minimal residual disease (MRD) by RT-PCR. However, after U-BMT it soon became undetectable. There was no detectable MRD until 7 months after U-BMT, but bone marrow relapse occurred 10 months after U-BMT. We consider that U-BMT is a promising treatment for t(16;21)(p11;q22) AML. However, an intensified conditioning regimen or modification of GVHD prophylaxis is needed.

Effect of chemotherapy and stem cell transplantation on T lymphocyte clones in familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare disorder in infancy, curative only by an allogeneic stem cell transplantation (SCT). We recently confirmed the clonal evidence of T cells in FHL. To confirm the effect of chemotherapy and SCT in FHL, the change of T-cell clones was analysed in two patients using inverse reverse transcription-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region (TCR V) gene, followed by PCR for the junctional region (Jbeta-PCR), a single-strand conformation polymorphism (SSCP) and sequencing analysis at diagnosis, after chemotherapy and after SCT. A high frequency (> 15%) of alphabeta T-cell clones and a predominant bias (Jbeta1:Jbeta2, 85:15) for the Jbeta1 subgroup were observed in the two patients at diagnosis. In one patient, however, an inverted predominant bias (Jbeta1:Jbeta2, 9:91) for the Jbeta2 subgroup and oligoclonal expansion were observed at relapse after chemotherapy. In the other patient, correction of both restricted Jbeta cluster usage and variation of TCR were observed after chemotherapy and SCT. Using sequence analysis, the clonal T cells detected at diagnosis were found to be substituted at low frequency (< 0.75%) by several new clones after chemotherapy and SCT. These results indicate that any genetic defect could influence the regulation of the T-cell network, and normalization of both the variation in each Vbeta repertoire and the Jbeta1/Jbeta2 ratio is needed to achieve remission, and might support the rationale that the only acceptable curative therapy of FHL is allogeneic SCT.

Requirement for etoposide in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

PURPOSE: We sought to identify the clinical variables most critical to successful treatment of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH). PATIENTS AND METHODS: Among the factors tested were age at diagnosis (< 2 years or > or = 2 years), time from diagnosis to initiation of treatment with or without etoposide-containing regimens, timing of cyclosporin A (CSA) administration during induction therapy, and the presence or absence of etoposide. RESULTS: By Kaplan-Meier analysis, the overall survival rate for the entire cohort of 47 patients, most of whom had moderately severe to severe disease, was 78.3% +/- 6.7% (SE) at 4 years. The probability of long-term survival was significantly higher when etoposide treatment was begun less than 4 weeks from diagnosis (90.2% +/- 6.9% v 56.5% +/- 12.6% for patients receiving this agent later or not at all; P <.01, log-rank test). Multivariate analysis with the Cox proportional hazards model demonstrated the independent prognostic significance of a short interval from EBV-HLH diagnosis to etoposide administration (relative risk of death for patients lacking this feature, 14.1; 95% confidence interval, 1.16 to 166.7; P =.04). None of the competing variables analyzed had significant predictive strength in the Cox model. However, concomitant use of CSA with etoposide in a subset of patients appears to have prevented serious complications from neutropenia during the first year of treatment. CONCLUSION: We conclude that early administration of etoposide, preferably with CSA, is the treatment of choice for patients with EBV-HLH.