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The risk of carcinogenesis increases after 20 years old in patients with Fanconi anemia (FA). We herein report three rare cases of FA combined with esophageal cancer in women; all patients were diagnosed with FA in early childhood. Patients 1 and 2 were diagnosed with advanced and superficial esophageal cancer, respectively, at 21 and 30 years old, respectively. Patient 3 was diagnosed with superficial esophageal cancer, underwent curative surgery at 26 years old, and survived for over 5 years without recurrence. Therefore, establishing a protocol for the early detection of esophageal cancer in FA patients over 20 years old is important.

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for juvenile myelomonocytic leukemia (JMML), but few large studies of HSCT for JMML exist. Using data from the Japan Society for Hematopoietic Cell Transplantation registry, we analyzed the outcomes of 129 children with JMML who underwent HSCT between 2000 and 2011. The 5-year overall survival (OS) rate and cumulative incidence of relapse were 64% and 34%, respectively. A regimen of busulfan/fludarabine/melphalan was the most commonly used (59 patients) and provided the best outcomes; the 5-year OS rate reached 73%, and the cumulative incidences of relapse and transplantation-related mortality were 26% and 9%, respectively. In contrast, the use of the irradiation-based myeloablative regimen was the most significant risk factor for OS (hazard ratio [HR], 2.92; P = .004) in the multivariate model. In addition, chronic graft-versus-host disease (GVHD) was strongly associated with lower relapse (HR, 0.37; P = .029) and favorable survival (HR, 0.22; P = .006). The current study has shown that a significant proportion of children with JMML can be cured with HSCT, especially those receiving the busulfan/fludarabine/melphalan regimen. Based on the lower relapse and better survival observed in patients with chronic GVHD, additional treatment strategies that focus on enhancing graft-versus-leukemia effects may further improve survival.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in thirteen cases from South Asia, twelve from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

Fanconi anemia is a rare recessive disease characterized by multiple congenital abnormalities, progressive bone marrow failure, and a predisposition to malignancies. It results from mutations in one of the 22 known FANC genes. The number of Japanese Fanconi anemia patients with a defined genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FANC genes in Japan and clarify the genotype-phenotype correlations. We studied 117 Japanese patients and successfully subtyped 97% of the cases. FANCA and FANCG pathogenic variants accounted for the disease in 58% and 25% of Fanconi anemia patients, respectively. We identified one FANCA and two FANCG hot spot mutations, which are found at low percentages (0.04-0.1%) in the whole-genome reference panel of 3,554 Japanese individuals (Tohoku Medical Megabank). FANCB was the third most common complementation group and only one FANCC case was identified in our series. Based on the data from the Tohoku Medical Megabank, we estimate that approximately 2.6% of Japanese are carriers of disease-causing FANC gene variants, excluding missense mutations. This is the largest series of subtyped Japanese Fanconi anemia patients to date and the results will be useful for future clinical management.

Fludarabine/cyclophosphamide-based conditioning regimens are standard in bone marrow transplantation (BMT) for acquired bone marrow failure in children, however, graft failure may occur. Using the data from a nationwide transplantation registry, we compared the outcomes of children aged

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by multiple congenital abnormalities, progressive bone marrow failure, and a predisposition to malignancies, resulting from mutations in one of the 22 known FANC genes (from FANCA to FANCW). The proteins encoded by these genes participate in a deoxyribonucleic acid interstrand cross-link repair pathway, the so-called FA/BRCA pathway. The 22 FANC genes include hereditary breast and ovarian cancer susceptibility genes, such as BRCA1 or BRCA2. Patients with FA display a wide range of clinical phenotypes owing to the genetic heterogeneity of the disease; therefore, the molecular diagnosis is critical for the appropriate management of such patients. Recently, we successfully subtyped 97% of the 117 Japanese patients with FA and identified 215 mutant alleles through a comprehensive strategy. In this review, the characteristics of genetic subtyping and mutated FANC gene variants in Japanese patients with FA and the genotype-phenotype correlation in FA are summarized. In addition, the carrier frequency of pathogenic FANC genes and risk of cancer among the FANC gene mutation carriers in general Japanese population are discussed.

Summary Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues., Graphical Abstract, Highlights • Toxic levels of genotoxic formaldehyde are produced endogenously in mammals • Two enzymes, ADH5 and ALDH2, are critical for clearance of endogenous formaldehyde • Their loss in mice and humans causes defective hematopoiesis and increased cancer • Elevated formaldehyde causes DNA damage and mutation signature found in many cancers, Dingler et al. show that formaldehyde is produced endogenously at sufficient levels to induce and overwhelm DNA repair. Two enzymes, ADH5 and ALDH2, are critical in clearance of formaldehyde, whose loss results in a bone marrow failure and leukemia syndrome of purely metabolic origin.

Mixed-phenotype acute leukemia (MPAL) is a rare type of leukemia expressing both myeloid and lymphoid markers. There is limited information, especially on pediatric cases. Therefore, the optimal therapeutic approach to pediatric MPAL has not been defined. Here, we report two pediatric cases of MPAL. According to the 2008 World Health Organization (WHO) classification and European Group for the Immunological Characterization of Leukemias (EGIL) criteria, patient 1 was diagnosed with overt MPAL positive for the myeloid marker myeloperoxidase (MPO), and B-lymphoid markers. Patient 2 was diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) using EGIL criteria. According to the 2008 WHO classification, however, patient 2 was diagnosed with overt MPAL positive for CD3, T-lymphoid markers and MPO. We chose an ALL-type therapy consisting of both lymphoid- and myeloid-directed agents; these patients have maintained complete remission following treatment. Further information on pediatric MPAL is needed to establish an appropriate therapeutic strategy including stem cell transplantation for this rare condition.

Studies using Fanconi anaemia (FA) mutant mouse models suggested that the combination of a defective FA pathway and aldehyde dehydrogenase-2 (ALDH2) dysfunction could provoke bone marrow failure, leukaemia and developmental defects, and that both maternal and fetal aldehyde detoxification are crucial to protect the developing embryo from DNA damage. We studied the ALDH2 genotypes of 35 Japanese FA patients and their mothers. We found that a normal maternal ALDH2 allele was not essential for fetal development of ALDH2-deficient patients, and none of the post-natal clinical parameters were clearly affected by the maternal ALDH2 genotype in these patients.

We report a case of HIV-negative Burkitt lymphoma (BL) that relapsed 9 years after complete remission. We performed a polymerase chain reaction analysis of three regions of the VDJ junction of the immunoglobulin heavy chain (IGH) gene and compared the clonality of the first and second BL lesions, which were found to be clonally distinct. The patient received the R-Hyper CVAD/R-MA regimen; however, leukoencephalopathy subsequently developed due to the effect of cytarabine, and the regimen was changed to R-IVAM. The patient achieved complete remission and received high-dose chemotherapy following autologous stem cell transplantation. He maintained the complete remission for 72 months after transplantation. Given this outcome, we suggest that clonally distinct relapse of HIV-negative BL may exhibit a good prognosis.

We retrospectively studied 108 marrow harvests from 105 pediatric sibling donors. The median age of donors was 8 years (range: 1-15) and the median body weight was 27 kg (range: 10-100). The volumes of aspirated marrow were 5.0-23.8 mL/kg donor body weight, and harvested bone marrow volume exceeded 15 mL/kg in 42% of the donors. A total of 100 autologous blood donations were performed, and eight donors had red cells salvaged from their harvests reinfused. The median Hb levels before and after harvests were 12.3 g/dL (range: 10.0-14.7) and 11.0 g/dL (range: 8.9-13.8), respectively. None of the donors received allogeneic blood transfusions or hematopoietic growth factors such as EPO and G-CSF before or after collection. Transplanted dose was 1.4-10.8 × 10(8) cells/kg recipient body weight without differences due to donor age. Higher concentrations of nucleated and CD34(+) cells were obtained from younger donors. All donors tolerated the procedures well, with no serious complications. Thus, children may safely donate marrow for allogeneic transplantation, and the yields of nucleated cells for engraftment are substantial.

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Precise genetic diagnosis of inherited bone marrow failure syndromes (IBMFS), a heterogeneous group of genetic disorders, is challenging but essential for precise clinical decision making. We analyzed 121 IBMFS patients using a targeted sequencing covering 184 associated genes and 250 IBMFS patients using whole-exome sequencing (WES). We achieved successful genetic diagnoses for 53 of 121 patients (44%) using targeted sequencing and for 68 of 250 patients (27%) using WES. In the majority of cases (targeted sequencing: 45/53, 85%; WES: 63/68, 93%), the detected variants were concordant with, and therefore supported, the clinical diagnoses. However, in the remaining 13 cases (8 patients by target sequencing and 5 patients by WES), the clinical diagnoses were incompatible with the detected variants. Our approach utilizing targeted sequencing and WES achieved satisfactory diagnostic rates and supported the efficacy of massive parallel sequencing as a diagnostic tool for IBMFS. Genet Med advance online publication 19 January 2017

Common variable immunodeficiency (CVID) is the most common adult-onset primary antibody deficiency disease due to various causative genes. Several genes, which are known to be the cause of different diseases, have recently been reported as the cause of CVID in patients by performing whole exome sequencing (WES) analysis. Here, we found FANC gene mutations as a cause of adult-onset CVID in two patients. B cells were absent and CD4+ T cells were skewed toward CD45RO+ memory T cells. T-cell receptor excision circles (TRECs) and signal joint kappa-deleting recombination excision circles (sjKRECs) were undetectable in both patients. Both patients had no anemia, neutropenia, or thrombocytopenia. Using WES, we identified compound heterozygous mutations of FANCE in one patient and homozygous mutation of FANCA in another patient. The impaired function of FANC protein complex was confirmed by a monoubiquitination assay and by chromosome fragility test. We then performed several immunological evaluations including quantitative lymphocyte analysis and TRECs/sjKRECs analysis for 32 individuals with Fanconi anemia (FA). In total, 22 FA patients (68.8%) were found to have immunological abnormalities, suggesting that such immunological findings may be common in FA patients. These data indicate that FANC mutations are involved in impaired lymphogenesis probably by the accumulation of DNA replication stress, leading to CVID. It is important to diagnose FA because it drastically changes clinical management. We propose that FANC mutations can cause isolated immunodeficiency in addition to bone marrow failure and malignancy.

Systemic mastocytosis (SM) associated with t(8;21) acute myeloid leukemia (AML) is very rare, and the D816 mutation of the KIT gene has previously been detected only in adult patients. We herein report the case of a 5-year-old female presenting with AML harboring t(8;21)(q22;q22). Her AML was refractory to chemotherapy, and bone marrow mastocytosis developed simultaneously at the initial diagnosis and during chemotherapy. The D816A mutation of KIT was detected. SM associated with t(8;21) AML, accompanied by a KIT mutation in children may result in a poor prognosis, despite the fact that t(8;21) AML are generally considered to have a favorable risk.

Fatty liver and male gonadal dysfunction are potential late effects of therapy in adult survivors treated with stem cell transplantation (SCT) in childhood. Obesity and metabolic syndrome also are associated with low serum testosterone levels in the general population. However, the relationship between the degree of fatty liver and changes in serum testosterone levels in adult survivors has not been fully studied. We reviewed the clinical records of 34 male patients who received allogeneic SCT in childhood or adolescence. The median age at SCT was 10.0 years, and the median follow-up after SCT was 15.9 years. All but one patient showed no tendency toward overweight/obesity during the follow-up period. Fatty liver was diagnosed by ultrasound in 15 patients at 4 to 20 years after SCT. Patients who received cranial radiation therapy before SCT were more likely to develop fatty liver and insulin resistance. Moreover, fatty liver was statistically associated with decreased serum testosterone levels, whereas nonfatty liver was not (median, 527 ng/dL [range, 168-944 ng/dL] versus 302 ng/dL [165-698 ng/dL]; P < .0001). Changes in testosterone levels after SCT are affected not only by primary gonadal dysfunction but also by subsequent development or exacerbation of fatty liver.

SCT from HLA-identical sibling donors is generally associated with an excellent survival in FA patients if performed prior to the development of MDS or leukemia. However, the optimal conditioning regimen has not been defined. We report here our experience with 15 Japanese FA patients who underwent HLA-matched sibling donor SCT. The aim of this study is to compare radiation-based conditioning to Flu-based conditioning for FA patients in a Japanese population where the T-cell somatic mosaicism is higher than in the Caucasian population. Eight patients (a-group) received a radiation-based conditioning (500-600 cGy of thoracoabdominal/TBI) with CY dose modification (20-120 mg/kg), and ATG; two patients exhibited rejection. Seven patients (b-group) received CY (40 mg/kg), 150-180 mg/m(2) of Flu, and ATG. Durable engraftment was demonstrated in all patients. In FA patients, Flu-based conditioning may allow stable engraftment in matched sibling donor transplantation without radiation, even in patients with T-cell somatic mosaicism.

Fanconi anemia is a rare autosomal recessive disease characterized by multiple congenital anomalies, pancytopenia, and cancer susceptibility, especially to leukemia and squamous cell carcinoma of the head and neck or esophagus. F-FDG PET/CT is a useful tool to assess tumor staging and follow-up of esophageal cancer. We report a rare case of cervical esophageal cancer and lymph node metastases detected on F-FDG PET/CT in a patient with Fanconi anemia after bone marrow transplantation.

Background Several studies have suggested that Langerhans cell histiocytosis (LCH) is responsive to treatment with bisphosphonates (BPs). However the efficacy and safety of BPs therapy for childhood LCH is unknown. Procedure Data on children with LCH who had received BPs therapy were collected retrospectively from hospitals participating in the Japanese Pediatric Leukemia/Lymphoma Study Group. Results Twenty-one children with histologically proven LCH were identified. Of these, the case histories of 16 children who had been treated with pamidronate (PAM) for disease reactivation were analyzed in detail. The median post-PAM therapy follow-up period was 2.8 years (range: 0.9–9.3 years). The median age at commencement of PAM therapy was 9.4 years (range: 2.3–15.0 years). All children had one or more bone lesions but none had risk organ (RO) involvement. In the majority of the children, six courses of PAM were administered at a dose of 1.0 mg/kg/course at 4-week intervals. In 12 of the 16 children, all active lesions including lesions of the skin (n = 3) and soft tissues (n = 3) resolved. Of these children, eight children had no active disease for a median of 3.3 years post-PAM therapy (range: 1.8–9.3 years). Progression-free survival (PFS) was 56.3 ± 12.4% at 3 years. PFS was significantly higher in children with a first reactivation compared with children experiencing a second or subsequent reactivation. Conclusions PAM may be an effective treatment for reactivated LCH with bone lesions. A prospective trial of the efficacy of PAM in recurrent pediatric LCH is warranted. Pediatr Blood Cancer. 2010;56:110–115. © 2010 Wiley-Liss, Inc.

Overweight/obesity among adult survivors of childhood SCT has been considered to be predictive of eventual development of metabolic abnormalities. Fatty liver is increasingly recognized as a major cause of liver-related morbidity and mortality in the general population. However, the real incidence of fatty liver in adult survivors of SCT has not been fully elucidated. We determined whether adult survivors are at risk for overweight/obesity, metabolic abnormalities and fatty liver and whether these risks are associated with cranial radiotherapy (CRT) before SCT. Among the 51 patients (30 males), only two male patients were overweight/obese at the last evaluation. On the other hand, 9 male (30%) and 15 female (71%) patients were underweight. Fatty liver was diagnosed in 11 male (37%) and 10 female (48%) patients during the follow-up period, although patients who had fatty liver did not tend to be overweight/obese. Significantly more patients who received CRT before SCT developed fatty liver with insulin resistance than those who did not (P

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

The number of long-term surviving stem cell transplant (SCT) recipients has increased steadily, and attention has now extended to the late complications of this procedure. The objective of this study was to investigate relationship among growth and endocrine functions in long-term adult survivors of childhood SCT. The inclusion criteria of this study were survival at least 5 yr after SCT and achievement of adult height. Fifty-four patients (39 males) fulfilled these criteria and were included in this study. Growth was mainly evaluated by height standard deviation score (SDS) and individual longitudinal growth curves. Among the 54 patients, those that received SCT before 10 yr of age showed significantly greater reductions in changes in height SDS (mean –1.75, range –4.80 to –0.10) compared with those that received SCT at or after 10 yr of age (mean –0.50, range –1.74 to 1.20; P

CCAAT/enhancer binding proteins (C/EBPs) have an important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid leukemia (AML). Their involvement in myelomonocytic leukemia, however, is still unclear. Therefore, the expression and function of C/EBPs in myelomonocytic cells with MLL-fusion genes were investigated. Retinoic acid (RA) induced monocytic differentiation in the myelomonocytic cell lines with MLL-fusion genes, THP-1, MOLM-14 and HF-6 cells, accompanied by monocytic differentiation with the upregulation of C/EBPα and C/EBPɛ. Monocytic differentiation by RA treatment was confirmed in primary AML cells using a clonogenic assay. When the activity of C/EBPα or C/EBPɛ was introduced into HF-6 cells, their cellular growth was arrested through differentiation into monocytes with the concomitant marked downregulation of Myc. Cebpe mRNA was upregulated by the induction of C/EBPα-ER, but not vice versa, thus suggesting that C/EBPɛ may have an important function in the differentiation process. Introduction of Myc isoforms into HF-6 cells partially antagonized the C/EBPs effects. These findings suggest that the ectopic expression of C/EBPɛ, as well as C/EBPα, can induce the monocytic differentiation of myelomonocytic leukemic cells with MLL-fusion gene through the downregulation of Myc, thus providing insight into the development of novel therapeutic approaches.

We report an 81-year-old man who had leukemic presentation of ALK-positive anaplastic large cell lymphoma (ALCL) as an initial manifestation. He had been well after chemotherapy and irradiation for the advanced lung adenocarcinoma, but suddenly suffered from severe lactic acidosis and hypotension. The peripheral blood smear and bone marrow aspiration revealed the infiltration of atypical large cells with horseshoe-shaped or lobulated nuclei. The detection of CD30 expression and the t (2;5) (p23;q35) translocation in these cells was confirmatory of a diagnosis of common variant ALK-positive ALCL in a leukemic phase. He deteriorated rapidly and died before administration of the chemotherapy. An adequate, prompt diagnosis is necessary for this rare disease status in oncologic emergency to improve the disease management.

Designing stem cell transplantation (SCT) conditioning regimens for Fanconi anemia (FA) has proved difficult because of hypersensitivity to the DNA cross-linking agents. We performed chromosome fragility tests with 56 FA patients and with 50 non-FA patients with severe aplastic anemia or myelodysplastic syndrome. We evaluated peripheral blood lymphocyte specimens cultured for 72 hours and treated with mitomycin C, diepoxybutane (DEB), cyclophosphamide (CY) metabolites, cytosine arabinoside (Ara-C), and fludarabine (Flu) metabolite (9-beta-D-arabinofuranosyl-2-fluoroadenine [2-F-Ara-A]). The DEB and CY metabolite tests were highly sensitive and specific for FA (P10(-4)) for both tests), and the number of aberrations per cell for DEB correlated with that for the CY metabolite test (P10(-4)) but did not correlate with the number of aberrations per cell for the Ara-C and 2-F-Ara-A tests. The difference in breakage frequencies between FA and non-FA patients for cultures treated with 2-F-Ara-A was not statistically significant. Most of the breakages observed in cells treated with 2-F-Ara-A-and Ara-C were chromatid breaks. It may be possible to determine the appropriate CY dose in the preconditioning regimen for SCT in FA patients on the basis of the in vitro effects on fragility, and Flu or Ara-C may be a safer drug than high-dose CY for conditioning in FA patients.

We evaluated the efficacy of treatment using reduced cumulative doses of anthracyclines in children with acute promyelocytic leukaemia (APL) in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-P05 study. All patients received two and three subsequent courses of induction and consolidation chemotherapy respectively, consisting of all-trans retinoic acid (ATRA), cytarabine and anthracyclines, followed by maintenance therapy with ATRA. Notably, a single administration of anthracyclines was introduced in the second induction and all consolidation therapies to minimize total doses of anthracycline. The 3-year event-free (EFS) and overall survival rates for 43 eligible children were 83·6% [95% confidence interval (CI): 68·6-91·8%] and 90·7% (95% CI: 77·1-96·4%), respectively. Although two patients died of intracranial haemorrhage or infection during induction phases, no cardiac adverse events or treatment-related deaths were observed during subsequent phases. Patients not displaying M1 marrow after the first induction therapy, or those under 5 years of age at diagnosis, showed inferior outcomes (3-year EFS rate; 33·3% (95% CI: 19·3-67·6%) and 54·6% (95% CI: 22·9-78·0%), respectively). In conclusion, a single administration of anthracycline during each consolidation phase was sufficient for treating childhood APL. In younger children, however, conventional ATRA and chemotherapy may be insufficient so that alternative therapies should be considered.

The inherited bone marrow failure syndromes (IBMFS) are rare disorders in which there is usually some form of bone marrow failure and typical changes in physical appearance, associated with a family history of the same disorder. Patients with IBMFS have a very high risk of developing myelodysplastic syndrome, acute myeloid leukemia, and solid tumors. The latest technology applied to the molecular pathogenesis of these disorders has led to identification of specific genetic mutations and now facilitates determining the appropriate diagnosis and management of afflicted patients. In this section, we describe physical and laboratory findings and management of the major IBMFS: Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, and Diamond Blackfan anemia. We also discuss their possible implications in the clinical features of Japanese patients.

Dyskeratosis congenita (DKC) is an inherited bone marrow failure (BMF) syndrome typified by reticulated skin pigmentation, nail dystrophy, and mucosal leukoplakia. Hoyeraal-Hreidarsson syndrome (HHS) is considered to be a severe form of DKC. Unconventional forms of DKC, which develop slowly in adulthood but without the physical anomalies characteristic of DKC (cryptic DKC), have been reported. Clinical and genetic features of DKC have been investigated in Caucasian, Black, and Hispanic populations, but not in Asian populations. The present study aimed to determine the clinical and genetic features of DKC, HHS, and cryptic DKC among Japanese patients. We analyzed 16 patients diagnosed with DKC, three patients with HHS, and 15 patients with cryptic DKC. We found that platelet count was significantly more depressed than neutrophil count or hemoglobin value in DKC patients, and identified DKC patients with large deletions in the telomerase reverse transcriptase and cryptic DKC patients with RTEL1 mutations on both alleles. This led to some patients previously considered to have unclassifiable BMF being diagnosed with cDKC through identification of new gene mutations. It thus seems important from a clinical viewpoint to re-examine the clinical characteristics, frequency of genetic mutations, and treatment efficacy in DKC, HHS, and cDKC.

Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF.

Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, increased cancer susceptibility, progressive bone marrow failure (BMF), and various developmental abnormalities resulting from the defective FA pathway. FA is caused by mutations in genes that mediate repair processes of interstrand crosslinks and/or DNA adducts generated by endogenous aldehydes. The UBE2T E2 ubiquitin conjugating enzyme acts in FANCD2/FANCI monoubiquitination, a critical event in the pathway. Here we identified two unrelated FA-affected individuals, each harboring biallelic mutations in UBE2T. They both produced a defective UBE2T protein with the same missense alteration (p.Gln2Glu) that abolished FANCD2 monoubiquitination and interaction with FANCL. We suggest this FA complementation group be named FA-T.

Fanconi anaemia (FA) is a genetically heterogeneous chromosome instability syndrome characterised by bone marrow failure and congenital anomalies. Although an increasing number of reports suggest that reversion mosaicism noted in peripheral blood lymphocytes (PBLs) is associated with mild haematopoietic failure in FA, myeloid cells are rarely directly examined. We here report a patient with prolonged mild pancytopenia in whom proliferation of revertant cells was detected in mature myeloid cells but not in PBLs. While this patient had inherited heterozygous mutations, 2546delC and 3720-3724del, in the major FA gene FANCA, Epstein-Barr virus-immortalised lymphoblastoid cells from the patient had 2546C > T instead of 2546delC, resulting in expression of a functional missense protein. As the identical reversion was detected in polymorphonuclear granulocytes and mononuclear phagocytes, sustained haematopoiesis in the patient can be attributed to a selective growth advantage of revertant myeloid cells. It is noteworthy that such a myeloid lineage-selective mosaicism is overlooked in routine examination of PBLs. Recognition of this status will expand the role of reversion mosaicism in the pathophysiology of FA.

An increasing number of long-term surviving bone marrow transplant (BMT) recipients have recovered from their primary disease but are at risk of developing failure of endocrine organs. We investigated 147 patients who underwent allogeneic BMT. Thyroid function was evaluated by serial measurement of basal TSH and free T4 levels as well as by TRH provocative test. Thyroid ultrasound examination was performed for evaluation of thyroid tumor after BMT. Five patients were found to have overt thyroid dysfunction (hypothyroidism in four patients and hyperthyroidism in one patient). Twenty-three patients in the under 9-yr-old group at BMT and 16 patients in the over 10-yr-old group at BMT had subclinical compensated hypothyroidism. Younger age at BMT was the strongest factor for developing thyroid dysfunction, compared with older age (P0.001). Only in patients with subclinical compensated hypothyroidism did median basal and peak TSH increase to the upper half of the normal range by 8 yr after BMT and then returned slightly to the middle of the normal range spontaneously. These results suggest that thyroid dysfunction in long-term BMT survivors depends on age at BMT, with a greater risk among younger patients, indicating the need for life-long surveillance.

To establish the most appropriate prophylactic therapy and risk factors for predicting hemorrhagic cystitis (HC) after stem cell transplantation (SCT), we retrospectively analyzed the clinical records of 450 transplant patients treated from 1982 to 2002. In all, 81 patients developed early- and/or late-onset HC (early=29, late=48, both=4). For the incidence of early-onset HC, administration of cyclophosphamide (CY) (p=0.0079, odds ratio (OD)=5.109, 95% confidence interval (CI)=1.533-17.030), busulfan (BU) (p=0.0015, OD=3.336, 95% CI=1.584-7.027), BU+CY (p=0.0001, OD=4.369, 95% CI=2.055-9.292), antithymocyte globulin (p=0.0009, OD=3.368, 95% CI=1.642-6.911), nonradiation (p=0.0163, OD=2.564, 95% CI=0.181-0.841), 2-mercaptoethane sodium sulfonate (Mesna) (p=0.0001, OD=7.519, 95% CI=2.847-19.858), and bladder irrigation (p=0.0001, OD=4.950, 95% CI=2.328-10.523) were risk factors. By Fisher's exact test, the combination of BU and Mesna was a more significant risk factor (P

BACKGROUND Familial hemophagocytic lymphohistiocytosis HLH (FHL) is fatal, unless patients are rescued with hematopoietic stem cell transplantation (SCT). Although the molecular identification of FHL now is possible at least in part from perforin gene study, many cases escape detection or never are tested due to the lack of specific hallmarks, making diagnosis difficult. To the authors' knowledge, it remains to be determined whether persistently low natural killer cell (NK) activity and a high incidence of central nervous system (CNS) disease increase the probability of FHL. METHODS The authors analyzed 42 HLH patients age < 2 years, 13 of whom developed overt CNS disease and 5 of whom demonstrated persistently deficient NK activity (Group 1). The remaining 24 patients had no CNS disease and had NK activity of moderate decrease to within the normal range (Group 2). RESULTS In Group 1, CNS symptoms were detected in 6 cases within 1 month and between 4.5–9 months in 6 other patients. In these cases, spotty lesions demonstrating a high T2 signal in the white matter were noted on brain magnetic resonance imaging. The survival was significantly poor for patients in Group 1 unless they were rescued with SCT, which was performed in 5 of the 13 patients with CNS disease and in all 5 patients with persistent NK activity deficiency. SCT was successful in 9 patients, with no CNS sequelae reported after the transplantation. Conversely, the prognosis of the 24 patients in Group 2 was better and only 1 patient required SCT. CONCLUSIONS Very young HLH patients (age < 2 years) who are at high risk of fatal FHL with persistently deficient NK activity and/or overt CNS disease require appropriate SCT to reverse CNS disease and achieve a complete cure. Cancer 2002;94:3023–31. © 2002 American Cancer Society. DOI 10.1002/cncr.10515

Lymphoid malignancies are rare in patients with Fanconi anemia (FA), particularly after bone marrow transplantation. A boy, who was diagnosed with FA at the age of 5; underwent successful bone marrow transplantation at the age of 11. One year later, he presented with fever and dry cough, and was found to have an anterior mediastinal tumor. Biopsy of the tumor revealed precursor-T cell lymphoblastic lymphoma. Human leukocyte antigen analysis confirmed that the tumor cells were derived from the patient's own cells. He received mild chemotherapy for lymphoma, but his condition deteriorated rapidly and he died from excessive chemotherapy-related toxicity. The literature contains no reports of successful chemotherapy for lymphoid tumors in patients with FA, and therefore, alternatives to chemotherapy should be considered in the treatment of such patients.

We report the outcomes of 30 patients with juvenile myelomonocytic leukemia (JMML) who received unmanipulated hematopoietic stem cell transplantation (HSCT) with oral or intravenous busulfan, fludarabine, and melphalan between 2001 and 2011. Mutations in PTPN11 were detected in 15 patients. Six patients received human leukocyte antigen (HLA)-matched HSCT from related donors, and 24 patients received HSCT from alternative donors, including 13 HLA-mismatched donors. Primary engraftment failed in five patients, all of whom had received allografts from HLA-mismatched donors. HLA-mismatched HSCT resulted in poorer event-free survival than HLA-matched HSCT (28.8 vs. 70.6 %). Three patients died of transplantation-related causes, and eight patients experienced hematological relapse (including five patients who died due to disease progression). Eight patients received a second HSCT, and four of these patients have survived. The 5-year estimated overall survival for all patients was 72.4: 88.9 % for the patients without a mutation in PTPN11 (n = 10) and 58.3 % for the patients with a mutation in PTPN11 (n = 15) (P = 0.092). The conditioning regimen reported in the present study achieved hematological and clinical remission in >50 % of patients with JMML who received HSCT from alternative donors, and may also be effective for JMML patients with PTPN11 mutation.

The clinical characteristics and prognostic relevance of acute myeloid leukaemia (AML) with myelodysplastic features remains to be clarified in children. We prospectively examined 443 newly diagnosed patients in a multicentre clinical trial for paediatric de novo AML, and found 'AML with myelodysplasia-related changes' (AML-MRC) according to the 2008 World Health Organization classification in 93 (21·0%), in whom 59 were diagnosed from myelodysplasia-related cytogenetics alone, 28 from multilineage dysplasia alone and six from a combination of both. Compared with 111 patients with 'AML, not otherwise specified' (AML-NOS), patients with 'AML-MRC' presented at a younger age, with a lower white blood cell count, higher incidence of 20-30% bone marrow blasts, unfavourable cytogenetics and a lower frequency of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), NPM1 and CEBPA mutations. Complete remission rate and 3-year probability of event-free survival were significantly worse in 'AML-MRC' patients (67·7 vs. 85·6%, P < 0·01, 37·1% vs. 53·8%, P = 0·02, respectively), but 3-year overall survival and relapse-free survival were comparable with 'AML-NOS' patients. By multivariate analysis, FLT3-ITD was solely associated with worse overall survival. These results support the distinctive features of the category 'AML-MRC' even in children.

The purified CD34+ cell fraction has been used for hematopoietic stem cell transplantation since they were demonstrated to have long-term reconstituting ability. Therefore, the potential effects of CD34− stem cells on the clinical course have been a major concern in recipients of CD34+-selected transplantation. To address this concern, we used an in vitro assay to determine whether transplant recipients have CD34−precursor population. Lin−CD34− cells were isolated from bone marrow cells in 11 transplant recipients including four CD34-selected transplantations, six standard bone marrow transplantations, and one T cell-depleted marrow transplantation. The frequency of the Lin−CD34− population in four CD34-enriched transplantation recipients was not different from those of normal donors or recipients of other modes of transplantation: 0.96 ± 1.01% (mean ± s.d., n = 4), 0.45 ± 0.16% (n = 6), and 0.66 ± 0.59% (n = 7), respectively. However, the Lin−CD34−population obtained from the recipients of CD34-enriched transplantation acquired neither CD34 expression nor colony-forming activity after 7 days of culture, whereas the cells from all the normal individuals and standard BMT recipients were able to differentiate into CD34+ cells accompanied by the emergence of colony-forming activity. We conclude that recipients of CD34-enriched transplantation appear to have defects in their CD34− precursor population. The clinical significance of these defects will be determined in a life-long follow-up of these patients. Bone Marrow Transplantation (2001) 28, 587–595.

Growth hormone (GH) deficiency has been regarded as a principal determinant for growth failure following bone marrow transplantation (BMT). We herein analyzed final height and GH secretion in the patients who received BMT during childhood. The study on final height in 30 patients (23 males; 19 with malignant disease) who underwent BMT before or at the onset of puberty showed the following findings: (1) Final height SD score (SDS) significantly decreased compared to pretreatment height SDS. (2) Patients who underwent BMT before the age of 10 years showed significantly greater reduction in height SDS compared to those who received after the age of 10 years. (3) The type of disease or a difference in preconditioning regimen did not influence the outcome of growth. (4) No patient showed GH deficiency. The study on GH secretion included 71 patients who had been followed for more than 5 years and who underwent insulin tolerance test more than twice following BMT. Thirteen patients experienced poor GH response at least once. Two of these patients had poor GH response repeatedly. In conclusion, children who undergo BMT at younger age have a higher risk of growth failure, and GH deficiency is not a major contributing factor for growth impairment following BMT.

Therapy-related myelodysplastic syndrome of recipient origin in a juvenile myelomonocytic leukemia patient 17 years after allogeneic BMT

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and a susceptibility to leukemia. There are at least 8 complementation groups (A through H). Extensive analyses of the FA group C gene FANCC in Western countries revealed that 10% to 15% of FA patients have mutations of this gene. The most common mutation is IVS4 + 4 A to T (IVS4), a splice mutation in intron 4, which has been found only in patients of Ashkenazi Jewish ancestry. When we screened 29 Japanese patients (20 unrelated patients and 4 families) using polymerase chain reaction-single strand conformation polymorphism, we found 8 unrelated patients homozygous for IVS4. This is apparently the first non-Ashkenazi-Jewish population for whom this mutation has been detected. The Ashkenazi Jewish patients homozygous for IVS4 have a severe phenotype, in comparison with other FA patients. Our analyses of Japanese patients indicate no significant difference between IVS4 homozygotes and other patients with regard to severity of a clinical phenotype. Thus, ethnic background may have a significant effect on a clinical phenotype in FA patients carrying the same mutation. (Blood. 2000;95:1493-1498)

The ability of mixed epidermal cell-lymphocyte reactions to detect allogeneic reactivities in an in vivo model was investigated by developing an in vivo model of acute graft-versus-host disease (GVHD), using SCID mice with a C.B-17 background in which human skin structures were generated by transplantation of cultured human epidermal cells (HEC) with dermal fibroblasts (HDFC). Suspensions containing cultured HEC and HDFC from a single donor were mixed with autologous peripheral blood mononuclear cells (PBMNC) or with PBMNC from unrelated individuals, and were injected into the flanks of C.B-17-SCID mice. Ten and 21 days after injection, subcutaneous nodules generated in the mice were examined histologically and immunohistochemically. Cystic structures developing after injection of HEC and HDFC without human PBMNC showed normal epidermislike tissue. Human skin generated in SCID mice injected with HEC and HDFC with auto-PBMNC showed no graft-versus-host reaction (GVHR) histologically, whereas those mice injected with PBMNC from siblings that shared an HLA haplotype showed mild GVHR. Human skin in SCID mice injected with HEC and HDFC with histoincompatible unrelated PBMNC showed moderate to severe GVHR. The severity of GVHR paralleled the dose of unrelated PBMNC, and GVHR was prevented by peroral treatment with cyclosporine A. Immunohistochemically, inflammatory cells infiltrating human cutaneous tissue formed in the SCID mice were stained by an anti-human CD45RO antibody that reacts with human T cells but not with murine lymphocytes, and most T cells were stained by an anti-human CD8 antibody recognizing HLA class I antigens. These findings are similar to those in clinical skin graft-versus host disease (GVHD) observed in patients undergoing allogeneic bone marrow transplantation. This experimental system should be useful as an in vivo model of human skin GVHD.

In a survey of childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) in Japan, we found 11p15 translocations in 5 (6%) of 81 children with t-AML/MDS. t(11;17)(p15;q21), t(11;12)(p15;q13), t(7;11)(p15;p15), inv(11)(p15q22), and add(11)(p15) were each found in one patient. Southern blotting and/or RT-PCR analyses revealed rearrangements of the NUP98 gene in tumor samples of all five patients. Rearrangements of DDX10 were detected in t-AML/MDS cells with inv(11), and rearrangements of HOXA9 were detected in t-AML cells with t(7;11). The 17q21 breakpoint of t(11;17) and the 12q13 breakpoint of t(11;12)(p15;q13) coincided with the loci of the HOXB and HOXC gene families, respectively. Therefore, it is reasonable to speculate that one of the HOXB genes and one of the HOXC genes were fused to NUP98 by t(11;17) and t(11;12), respectively, in t-AML/MDS cells. We propose that NUP98 may be a target gene for t-AML/MDS, and that t-AML/MDS with a fusion of NUP98 and HOX or DDX10 genes may be more frequent in children than in patients of other age groups.

Interleukin-12 (IL-12) is a crucial cytokine regulating cell-mediated immunity, and may contribute to the development of graft-versus-host disease (GVHD). We investigated serum IL-12 concentrations, interferon gamma (IFN-gamma) production by peripheral blood lymphocytes (PBL) from allogeneic stem cell recipients after IL-12 plus anti-CD3 monoclonal antibody (mAb) stimulation. We also investigated IL-12 production by peripheral macrophages (Mphi) after lipopolysaccharide (LPS) stimulation from allogeneic stem cell recipients and patients receiving donor leukocyte transfusions (DLT) for treatment or prophylaxis of leukemia relapse and Epstein-Barr virus (EBV) lymphoproliferative disease (LPD). PBL from acute GVHD patients produced high IFN-gamma levels after IL-12 plus anti-CD3 mAb stimulation, whereas PBL from patients without acute GVHD produced low levels of IFN-gamma. However, serum IL-12 concentrations were low in both groups. Peripheral Mphi IL-12 production increased in patients who developed acute GVHD compared to patients without acute GVHD. Five patients receiving DLT for treatment or prophylaxis of leukemia relapse developed acute GVHD. IFN-gamma production by PBL stimulated by IL-12 plus anti-CD3 mAb increased, while IL-12 production by peripheral Mphi stimulated by LPS was very high after the development of acute GVHD. However, serum IL-12 concentration remained low. Three patients receiving DLT for EBV-LPD did not develop acute GVHD with no increase of IFN-gamma and IL-12 production. These results indicate that IL-12 may play an important role in the development of acute GVHD after allogeneic stem cell grafting or DLT, and increased IL-12 production by Mphi occurs with various stimuli, including LPS.

Fanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach., 「アルデヒド分解酵素遺伝子」ALDH2が解き明かす難病の謎. 京都大学プレスリリース. 2013-09-13