Mikiya Endo

Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles Quantification Using Neonatal Guthrie Cards

OBJECTIVE To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION TRECs quantification can be used as a neonatal mass screening for patients with SCID.

Hypercalcemia in childhood acute lymphoblastic leukemia: frequent implication of parathyroid hormone-related peptide and E2A-HLF from translocation 17;19.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (>12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (<20 × 109/l), 15 showed hemoglobin⩾8 g/dl and 14 showed platelet count ⩾100 × 109/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Functional analysis of JAK3 mutations in transient myeloproliferative disorder and acute megakaryoblastic leukaemia accompanying Down syndrome

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS‐AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS‐AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient‐derived JAK3 mutation (JAK3I87T), as well as two novel JAK3 mutations (JAK3Q501H and JAK3R657Q) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof‐of‐principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS‐AMKL patients carrying activating JAK3 mutations.

Quantitative assessment of PTPN11 or RAS mutations at the neonatal period and during the clinical course in patients with juvenile myelomonocytic leukaemia.

To evaluate minimal residual disease (MRD) after chemotherapy and haematopoietic stem cell transplantation in juvenile myelomonocytic leukaemia (JMML), a locked nucleic acid‐allele specific quantitative polymerase chain reaction (LNA‐AS‐qPCR) was developed for 13 patients (four types of PTPN11 mutation and four types of RAS mutation). The post‐transplant MRD detected by LNA‐AS‐qPCR analysis was well correlated with chimerism assessed by short tandem repeat PCR analysis. Non‐intensive chemotherapy exerted no substantial reduction of the tumour burden in three patients. There was no significant difference in the quantity of RAS mutant DNA after spontaneous haematological improvement in 4 patients with NRAS or KRAS 34G > A during a 2‐ to 5‐year follow‐up. PTPN11, NRAS, or KRAS mutant DNA was detected from Guthrie card dried blood in five of seven patients (who were aged <2 years at diagnosis) at a level of 1·0–6·5 × 10−1 of the values at diagnosis. Accordingly, these five patients might have already reached a subclinical status at birth. Considering the negative correlation between mutant DNA level in neonatal blood spots and age at diagnosis, JMML patients with a larger tumour burden at birth appeared to show earlier onset.

The key role of stem cell factor/KIT signaling in the proliferation of blast cells from Down syndrome-related leukemia.

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

Naturally occurring oncogenic GATA1 mutants with internal deletions in transient abnormal myelopoiesis in Down syndrome

Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.

Correlation of chromosome abnormalities with presence or absence of WT1 deletions/mutations in Wilms tumor.

Of 40 Wilms tumors with chromosome abnormalities, 6 were hypodiploid, 10 were pseudodiploid, 7 were hyperdiploid with 47 to 49 chromosomes, and 17 were hyperdiploid with 50 or more chromosomes, mostly including +12. WT1 deletions/mutations were found in one hypodiploid, eight pseudodiploid, and one hyperdiploid (47–49 chromosomes) tumor, but in none of the hyperdiploid (≥50 chromosomes) tumors. Of the 10 tumors with WT1 abnormalities, 6 had a homozygous WT1 deletion, 1 had a nonsense WT1 mutation and loss of heterozygosity at 11p, 1 had an intragenic hemizygous WT1 deletion without detectable WT1 mutation, and 2, which occurred in Wilms tumor–aniridia–genitourinary abnormalities–mental retardation syndrome patients, had a hemizygous deletion and a missense or frameshift mutation of WT1. Six of the nine tumors with homozygous or hemizygous WT1 deletions had chromosome aberrations involving chromosome band 11p13 in one of the two chromosomes 11. While one hypodiploid and one pseudodiploid patient died of the disease, and one hyperdiploid (47–49 chromosomes) patient was alive in nonremission, all hyperdiploid (≥50 chromosomes) patients had no evidence of disease at the last follow‐up. Our data show that chromosome aberrations are closely correlated to WT1 abnormalities and suggest that hyperdiploid (≥50 chromosomes) Wilms tumors may be characterized by the absence of WT1 abnormalities and possibly also by a favorable prognosis. Genes Chromosomes Cancer 25:26–32, 1999.

Quantitative Assessment of Minimal Residual Disease in Childhood Lymphoid Malignancies Using an Allele-Specific Oligonucleotide Real-Time Quantitative Polymerase Chain Reaction

We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/105 cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lym-phoblastic leukemia and 3 of non-Hodgkin’s lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.

Upper gastrointestinal disorders induced by non-steroidal anti-inflammatory drugs

Abstract.Aims:We examined the characteristics of upper gastrointestinal disorders induced by non-steroidal anti-inflammatory drugs (NSAIDs).Methodology:The questionnaire investigation was performed over a five year period.Results:A study was performed on 354 patients (161 men and 193 women with mean ages of 66.0 and 70.7 years, respectively) who developed NSAIDs associated upper GI disorders: 21 patients had AGML, 212 had gastric ulcer, 63 had duodenal ulcer, 17 had gastroduodenal ulcers and 41 other cases. About 75 % of patients received NSAIDs for orthopedic conditions. Sixty percent of gastric disorders induced by NSAIDs affected the antrum or angulus of the stomach. The incidence of disorders of the gastric antrum was significantly higher in women than in men whilst the incidence of disorders on the gastric angulus was significantly higher in men than in women (p < 0.05). The proportion of patients with abdominal pain was significantly lower in patients over 65 years old than in those under 65 years old, and the proportion of patients with hematemesis or melena was significantly higher in patients over 80 years old than in those under 80 years old (p < 0.05). The time taken to achieve the healing stage was significantly longer in patients with greater than 3 months NSAIDs ingestion compared to patients that had received NSAIDs for less than 3 months (p < 0.05).Conclusions:Patients 65 years old and over with continuous NSAIDs use had asymptomatic ulcers, and patients 80 years old and over had hemorrhagic ulcers.