Kana Washio

Multi-Step Aberrant CpG Island Hyper-Methylation Is Associated with the Progression of Adult T–Cell Leukemia/Lymphoma

Aberrant CpG island methylation contributes to the pathogenesis of various malignancies. However, little is known about the association of epigenetic abnormalities with multistep tumorigenic events in adult T cell leukemia/lymphoma (ATLL). To determine whether epigenetic abnormalities induce the progression of ATLL, we analyzed the methylation profiles of the SHP1, p15, p16, p73, HCAD, DAPK, hMLH-1, and MGMT genes by methylation specific PCR assay in 65 cases with ATLL patients. The number of CpG island methylated genes increased with disease progression and aberrant hypermethylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival by Kaplan-Meyer analysis. The present findings strongly suggest that the multistep accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression, and prognosis of ATLL, as well as indicate the value of CpG methylation and CIMP for new diagnostic and prognostic biomarkers.

Activation of Akt is Associated With Poor Prognosis and Chemotherapeutic Resistance in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Activation of the phosphoinositide 3‐kinase (PI3K)/Akt pathway, a pro‐survival pathway, plays important roles in tumor cell growth. However, the role of Akt in the pathogenesis of pediatric B‐precursor acute lymphoblastic leukemia (B‐pre ALL) remains to be clarified. This study was undertaken to explore the clinical relevance and molecular mechanisms underlying the activation of Akt (i.e., phosphorylated Akt, P‐Akt) in pediatric B‐pre ALL.

Two cases of chronic active Epstein-Barr virus infection in which EBV-specific cytotoxic T lymphocyte was induced after allogeneic bone marrow transplantation.

Abstract:  CAEBV is a high mortality and morbidity disease with life‐threatening complications. Nevertheless, the treatment regimens for CAEBV have not yet been established. Although some reports have described CAEBV therapy involving treatments such as antiviral drugs, immunomodulatory agents, and immunochemotherapy, none of these treatments have been demonstrated to be effective. The only treatment reported to be effective is allogeneic SCT. However, the complications of SCT are severe, so treatment results have been poor. Recently, immunotherapy has been devised, but this is still in the developmental stage. In this report, two cases of CAEBV in which allogeneic SCT was performed soon after diagnosis are reported. In both cases, a high EBV genome titer in the peripheral blood was detected at onset. After SCT, the EBV genome titer decreased as CTL activity gradually increased. This fact suggested that not only high‐dose chemotherapy as a preconditioning treatment of SCT but also increased CTL activity which could eliminate virus‐infected cells might be effective, although additional cases should be studied in order to establish effective treatments.

ABL kinase mutation and relapse in 4 pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia cases

The tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL), which had showed poor prognosis before the dawn of IM treatment. However, if Ph-ALL patients showed IM resistance due to ABL kinase mutation, second-generation TKI, dasatinib or nilotinib, was recommended. We treated 4 pediatric Ph-ALL patients with both IM and bone marrow transplantation (BMT); however, 3 relapsed. We retrospectively examined the existence of ABL kinase mutation using PCR and direct sequencing methods, but there was no such mutation in all 4 diagnostic samples. Interestingly, two relapsed samples from patients who were not treated with IM before relapse did not show ABL kinase mutation and IM was still effective even after relapse. On the other hand, one patient who showed resistance to 3 TKI acquired dual ABL kinase mutations, F359C at the IM-resistant phase and F317I at the dasatinib-resistant phase, simultaneously. In summary, Ph-ALL patients relapsed with or without ABL kinase mutation. Furthermore, ABL kinase mutation was only found after IM treatment, so an IM-resistant clone might have been selected during the IM treatment and intensive chemotherapy. The appropriate combination of TKI and BMT must be discussed to cure Ph-ALL patients.

Relapsed infant MLL-rearranged acute lymphoblastic leukemia with additional genetic alterations

Acute lymphoblastic leukemia (ALL) in infants is a rare and aggressive disease, and has poor prognosis. Approximately 80% of patients with infant ALL harbor a mixed lineage leukemia (MLL, also known as KMT2A) gene rearrangement, which is a strong independent predictor of poor outcome.1,2 Although MLL rearrangement is considered a strong driver mutation, it may not be sufficient for leukemogenesis.3 We report two relapsed cases of infant ALL with MLL rearrangement. Case 1 is a 3-month-old female who was diagnosed as infant ALL with MLL-ENL, and Case 2 is a 4-month-old male who was diagnosed as infant ALLwithMLL-AF4. Both patients experienced a relapse twice. Somatic DNA was obtained from bone marrow at diagnosis and each episode of relapse, while germline DNA was obtained from buccal swab. Targeted sequencing was performed on MiSeq (Illumina, San Diego, CA) using HaloPlex custom panels (Agilent, Santa Clara, CA), with 163 known mutated genes in hematological diseases, which are customized to detect cancer-related genes (Supplementary Table S1). Read alignment and variant calling were performed using the MiSeq reporter (Illumina), and variants were annotated using SureCall software (Agilent). Synonymous or noncoding variants were excluded, and single nucleotide polymorphisms (SNPs) and germline polymorphisms were reported to dbSNP, unless they were found in the COSMIC database. Among the remaining variants with a variant allele frequency higher than 0.1, recurrentlymutated genes reported in a larger cohort4 were considered as candidate genes and validated by conventional Sanger sequencing. Sanger sequencingwasperformedafterPCR using specific primers, and mutations were detected by ABI PRISM 3100 (Thermo Fisher Scientific,Waltham,MA) using the primers listed in Supplementary Table S1. Thus, we identified additional gene alterations (Fig. 1). Only two mutations were detected in diagnostic samples, indicating that small number of mutations cooperate with MLL rearrangement. Among them, mutations in KRAS and FLT3 have also been recurrently reported in a study by Andersson et al. using wholegenome or whole-exome sequencing .4 To date, their study has been the largest describing the mutational profile of infant ALL. It is notable that many mutations other than those involved in the tyrosine kinasePI3K-RAS signaling pathways, which are found in approximately half of the patients, are found with low frequency. We could monitor our

Successful treatment of unresectable advanced hepatoblastoma: Living liver transplantation after surgical removal of lung metastasis

Miyamura T, Yoshida R, Yagi T, Matsukawa H, Chayama K, Ishida T, Washio K, Morishita N, Oda M, Morishima T. Successful treatment of unresectable advanced hepatoblastoma: Living liver transplantation after surgical removal of lung metastasis.
Pediatr Transplantation 2011: 15: E87–E91.

Adults with germline CBL mutation complicated with juvenile myelomonocytic leukemia at infancy

Juvenile myelomonocytic leukemia (JMML) appears to be a life-threatening disease and showed poor prognosis even after hematopoietic stem cell transplantation (HSCT) because of high relapse rate. On the other hand, recent molecular analysis revealed the heterogeneity of JMML. Here we report that two JMML patients survived >20 years without HSCT and both patients had uniparental disomy of 11q23 where CBL is located without the phenomenon found in neither Noonan syndrome nor Noonan syndrome-like disorder. We think that some JMML patients with CBL mutation might show the good prognosis in later life after remission of JMML.

Gene expression analysis of hypersensitivity to mosquito bite, chronic active EBV infection and NK/T-lymphoma/leukemia

Abstract The human herpes virus, Epstein-Barr virus (EBV), is a known oncogenic virus and plays important roles in life-threatening T/NK-cell lymphoproliferative disorders (T/NK-cell LPD) such as hypersensitivity to mosquito bite (HMB), chronic active EBV infection (CAEBV), and NK/T-cell lymphoma/leukemia. During the clinical courses of HMB and CAEBV, patients frequently develop malignant lymphomas and the diseases passively progress sequentially. In the present study, gene expression of CD16(−)CD56(+)-, EBV(+) HMB, CAEBV, NK-lymphoma, and NK-leukemia cell lines, which were established from patients, was analyzed using oligonucleotide microarrays and compared to that of CD56brightCD16dim/− NK cells from healthy donors. Principal components analysis showed that CAEBV and NK-lymphoma cells were relatively closely located, indicating that they had similar expression profiles. Unsupervised hierarchal clustering analyses of microarray data and gene ontology analysis revealed specific gene clusters and identified several candidate genes responsible for disease that can be used to discriminate each category of NK-LPD and NK-cell lymphoma/leukemia.

Persistent clonal chromosomal abnormalities in a chronic myeloid leukemia patient

Clonal cytogenetic abnormalities (CCA) in Philadelphia chromosome (Ph)‐negative cells have been reported in a small population of adult chronic myelogenous leukemia (CML) patients during the clinical course, but CCA in pediatric CML patients are rarely reported. We herein report the case of an 8‐year‐old boy from the onset of CML. Although he had relapse after unrelated bone marrow transplantation when 9 years old, he has since been in complete molecular response on imatinib mesylate treatment. Surprisingly, various CCA have been observed in this patient, including several reciprocal chromosomal translocations in Ph‐negative cells for >12 years. Although dysplasia in the bone marrow cells was identified, no overt transformation to myelodysplastic syndrome or acute myeloid leukemia has been observed. The cause of the CCA remains unknown in this patient, and careful observation is required.

Cumulative kinetics of epigenetic abnormalities during initiation and progression of Adult T-cell Leukemia/Lymphoma (ATLL)

HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years. ATLL is divided into four stages: namely, smoldering, chronic, lymphoma and acute types. The smoldering and chronic types are indolent, but the acute and lymphoma types are aggressive ATLL characterized by resistance to chemotherapy and a poor prognosis. Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical events in the progression have not been characterized. To determine whether epigenetic abnormalities are playing important roles in the progression of ATLL, we analyzed the methylation profiles, showing that number of CpG island methylated genes increased with disease progression and aberrant hyper-methylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival with the Kaplan-Meyer analysis. Increase of aberrant DNA methylation density was observed during the progression of an ATLL patient. The present findings strongly suggest that the multi-step accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the initiation and progression of ATLL not only epidemiologically but also in the clinical course of a specific ATLL patient.