京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22152号 / 社医博第100号 / 新制||社医||10(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 川上 浩司, 教授 武藤 学, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23101号 / 医博第4728号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 松田 文彦, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La cancérogénicité du benzène à forte dose (CIRC, Groupe 1) a été démontrée sur la base des cas de leucémies observés dans les études épidémiologiques conduites en milieu professionnel chez l’adulte exposé à de fortes doses. Ce polluant est émis en faible concentration dans l’environnement, notamment par le trafic routier et les stations-service. L’objectif est d’étudier le lien entre exposition au benzène à faible dose et risque de leucémie chez l’enfant. L’étude GEOCAP regroupe les cas de leucémies de l’enfant diagnostiqués en France entre 2002 et 2007 issus du Registre National des Hémopathies de l’Enfant, et un échantillon de 30 000 témoins représentatif de la population générale pédiatrique. La longueur de routes à fort trafic a été calculée au voisinage de la résidence de chaque sujet géocodé dans un rayon de 150 m. Les concentrations en benzène et en NO2 ont été estimées pour l’ensemble des sujets de France continentale à une échelle de 2 km2. La distance à la station-service la plus proche a également été calculée depuis la résidence de chaque sujet. La longueur de routes au voisinage de la résidence était positivement et significativement associée au risque de leucémie aigüe myéloblastique (LAM). La prise en compte des expositions au benzène et en NO2 renforçait cette association. La distance à la station-service la plus proche était positivement mais non-significativement associée à la LAM, et cette association résultait en partie d’un effet de confusion de la longueur de routes au voisinage de la résidence. Ce travail soutient l’hypothèse selon laquelle l’exposition au benzène émis dans l’environnement pourrait induire une augmentation du risque de LAM chez l’enfant. / In adults, the relationship between benzene exposure (IARC, Group 1) and leukemia has been demonstrated in workplace for high-level exposure. This pollutant is emitted at low concentration in environment, especially by road traffic and petrol stations. The objective was to investigate the relationship between benzene exposure at low-level dose and childhood leukemia. The record-based GEOCAP study included all cases of childhood leukemia diagnosed in France over 2002-2007 and 30,000 contemporaneous population-based control children highly representative of the source population. The length of major roads was calculated around the residence of each geocoded subject in a 150-m buffer. We assigned them the yearly estimates of benzene and NO2 concentrations at the square where his residence was located on a 2-km2 grid covering continental France. The distance to the nearest petrol station from the residence was also calculated for all subjects. The length of major roads around the residence was positively and significantly associated with the risk of acute myeloblastic leukemia (AML). This association was more evident with benzene and NO2 2-km estimates, combined with the length of major roads indicator to enhance the exposure contrasts. The distance to the nearest petrol station was positively but non-significantely associated with AML, and some of this association was explained by a confouding effect of the length of major roads around the residence. This work suggests that exposure to benzene emitted in environment may increase the risk of AML in children.

This study examines the risk of cancer in children of pesticide applicators from the Agricultural Health Study. The study includes 36,537 children of Iowa participants who were evaluated for cancer incidence during 1975 through 2013 from birth through the age of seventeen. Standard incidence rates for any cancer and specific groups of cancers classified using the International Classification of Childhood Cancer was calculated using rates from the general population of Iowa controlling for year of follow, age, sex, and race. Hazard ratios for Group I-III cancers and paternal exposure to specific pesticides were calculated using exposure information collected on 50 pesticides during phase 1 and 2 of the Agricultural Health Study. The exposure information allowed for calculation of intensity-weighted days of exposure to pesticides using the Agricultural Health Study exposure algorithm. Additionally, maternal ever exposure to specific pesticides was used to evaluate risk of childhood cancer. There were 118 cancers identified in children of Agricultural Health Study participants. The all-cancer standardized incidence ratio was significantly elevated (SIR = 1.27 95% CI: 1.04-1.50). The most common groups of cancers were Group I leukemia, myeloproliferative disease, and myelodysplastic disease (n=34) followed by Group III central nervous system (CNS) and miscellaneous intracranial and intraspinal neoplasms (n=25). For paternal intensity-weighted days of exposure, there were 31 of 50 specific pesticides that had sufficient cases of cancer to investigate using Cox proportional hazard models. The herbicide trifluralin significantly increased the risk for Group I childhood cancers for any parental pesticide exposure 2 years before birth through birth when compared to children with no paternal exposure (HR = 2.72 95% CI: 1.15, 6.44). This was consistent with results found from analyzing exposure split into two quantiles based on median exposure of exposed children with a Group I cancer. Parental use of the herbicide S-Ethyl-dipropylthiocarbamate (EPTC) did not result in a sufficient number of Group III cancer cases to look at levels of exposure to EPTC, but ever exposure showed an increased hazard ratio when compared to children with unexposed fathers (HR = 2.56 95% CI: 1.06, 6.20). Other pesticides (dicamba, cyanazine, and terbufos) showed mixed evidence of an association with specific childhood cancers, but were either under powered to evaluate with sensitivity analysis or showed inconsistent risk across exposure levels. Less extensive exposure information was available for mothers of children of the Agricultural Health Study, so analysis was restricted to ever or never exposure to pesticides during a mother’s lifetime. Additionally, there were a limited number of cases of cancer for which maternal exposure to specific pesticides was reported resulting in only 4 pesticides being evaluated for childhood cancer risk (glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), carbaryl, and malathion). For these four pesticides, this study did not detect any increased risk of childhood cancer from maternal exposure. In summary, this study provides the first epidemiological evidence of an increased risk of childhood cancer for trifluralin and EPTC. Since this study provides the first evidence of this increased risk, additional analysis is needed to validate the results. This study demonstrates how pesticide exposure information from participants of the AHS can be used in the evaluation of their children’s cancer risk. Additional follow-up and analysis of this cohort beyond the age of 17 would provide further insight into cancer risk during early adulthood from early life pesticide exposure.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by great heterogeneity and clonal nature. In recent years, rapidly evolving next-generation sequencing methods provided a deep insight into the mutational background of AML. It was shown that ~ 44 % of AML patients harbor mutations in genes that regulate DNA methylation. So far, many researchers have tried to evaluate the prognostic significance of DNA methylation changes in AML, however, due to a great inconsistency in these studies, none of the reported markers were implemented into clinical practice. The aim of this work was to further investigate the DNA methylation changes in AML patients with specific mutations and their prognostic effect. Next, we wanted to develop a new approach for a complex evaluation of prognostically significant DNA methylation aberrations. In our first project, we assessed the overall DNA methylation, hydroxymethylation, and gene expression in AML patients with mutations in either DNMT3A or IDH1/2 or their combinations. We discovered that each genetic aberration is connected with a distinct pattern of DNA hydroxy-/methylation changes that are not entirely reflected in altered gene expression. Patients with mutations in both genes exhibited a mixed DNA methylation profile most similar to healthy...

CCAAT/Enhancer Binding Protein Alpha (C/EBPa) is transcription factor protein involved in the differentiation of many cell types, including granulocytes and pulmonary cells. Studies have found that downregulation of C/EBPa leads to tumor formation in the hematopoietic system, bones, lungs, liver, and other organs. Mutations and post translational modifications can also reduce the function of C/EBPa in humans, leading to cancers. Recent studies have made progress in treating acute promyelocytic leukemia (APL), an M3 subtype of acute myeloid leukemia (AML), by incorporating all trans retinoic acid (ATRA) in targeted treatments. ATRA increases C/EBPa expression levels, thus promoting cell differentiation and subsequent apoptosis of leukemia cells. Still, survival rates of AML patients are low. In patients diagnosed with AML subtypes M4 or higher, ATRA does not work. In addition, patients can become resistant to ATRA, making it essential to find an alternative therapy. Therefore, novel drug treatments are necessary. Through a high throughput screening method, we have determined a potent chemical compound, ICCB280, that can enhance C/EBPa expression levels. However, ICCB280’s effective concentration for cell differentiation is relatively high, so we performed a structural-activity relationship (SAR) analysis and discovered a more potent chemical, styryl quinazolinone CCAAT/Enhancer Binding Protein Compound 73 (CEBP- 73). We tested CEBP-73 with two cell lines, HL-60 and A549, which represent leukemia and lung cancer models, respectively. We found that CEBP-73 increased C/EBPa expression levels in a time-dependent, dose-responsive manner in both leukemia cells and lung cancer cells. In western blot analyses, while both ICCB280 and CEBP-73 upregulated C/EBPa protein expression, more protein was expressed in leukemia and lung cancer cells treated with CEBP-73 in a dose-dependent manner than in cells treated with ICCB280. Next, we investigated CEBP-73’s effectiveness in upregulating C/EBPa’s downstream genes. We observed enhanced expression of CEBPe (HL-60 specific downstream gene) in HL-60 cells, and enhanced SPC, NKX2-1 (codes for TTF-1), and HIF-1a (A549 specific downstream genes) expression levels in A549 cells. To investigate the mechanisms of increased C/EBPa expression, we asked whether expression of an extra coding CEBPA (ecCEBPA), a noncoding RNA for C/EBPa that prevents methylation at the CEBPA gene promoter site, will increase. We found that CEBP-73 increased not only C/EBPa expression, but also ecCEBPA in HL-60 and A549 cells. This is the first study to our knowledge that confirms styryl quinazolinone CEBP- 73 can increase ecCEBPA expression. To examine the effectiveness of CEBP-73 in vivo, EGFR-L858R-T790M (EGFRTL/CCSP-rtTA) mice were administered a vehicle solution (control), 1 mg/kg of CEBP-73, or 10 mg/kg CEBP-73. The results showed a trend in CEBP-73 concentrations; higher doses of CEBP-73 induce higher levels of C/EBPa expression in lung tissue. Fewer and smaller tumors were present in lungs treated with CEBP-73 than lungs treated with a control. These findings support the role of CEBP-73 in enhancing C/EBPa expression, including upregulation at the promoter region of the CEBPA gene and at downstream gene loci. In addition, the study’s results affirm the role of C/EBPa as an inducer of cell differentiation in leukemia and lung cancer by showing neutrophils with segmented lobes and granules, indications of cell maturity, in cells treated with compounds that enhanced C/EBPa expression. These data suggest that CEBP-73 could provide novel therapeutic approaches in treating leukemia and lung cancer and could potentially be modified to treat other cancers in targeted drug therapies.

Non-Secretory Multiple Myeloma (NSMM) is characterized by typical morphological and pathological multiple myeloma (MM) characteristics and the absence of an M-protein on immunofixation electrophoresis with estimated prevalence of 3%. Among the NSMM cases there is a subset in which no cytoplasmic Immunoglobulin synthesis is detected, and this entity is called ‘’Non-Producing’’ Multiple Myeloma (NPMM). Plasma cell leukemia (PCL) is an aggressive form of MM characterized by high levels of abnormal plasma cells circulating in the peripheral blood. We present a rare case of non-producing variant of PCL. 75-year-old male was admitted due to anemia and thrombocytopenia. His CBC revealed hemoglobin of 9.0 g/dl and platelets were 9 k/ul. CMP showed creatinine of 1.34 mg/dl, total protein of 6 g/dl, albumin 3.6 g/dl and corrected calcium was normal. LDH was 204 IU/L. Peripheral smear review showed 8% circulating atypical plasmacytoid cells, normochromic normocytic anemia and thrombocytopenia. SPEP showed no monoclonal protein. IgA was normal. IgG, IgM were low 315 mg/dl and 20 mg/dl respectively. Serum beta-2 microglobulin was high (5.5, 1.1 – 2.4 mg/dl). Serum free kappa light chain was low (0.15, 0.33-1.94 mg/dl), lambda light chain and ratio was normal. Skeletal survey showed possible lytic lesions in right femur neck and subtrochanteric left femur. Bone marrow biopsy showed plasma cell myeloma involving 90-95% of bone marrow cellularity. The plasma cells show morphologic heterogeneity with prominent immature, plasmablastic and pleomorphic morphology. Flow cytometry shows a dominant abnormal CD45-dim population with expression of CD38, CD138, CD56 and CD117 (partial). The abnormal cells are negative for cytoplasmic kappa and lambda immunoglobulin light chains and negative for myeloid and lymphoid markers (by flow cytometry and immunohistochemical stains). Complex chromosomal analysis. Plasma cell FISH studies was positive for t(11;14). Based on suggested revised diagnostic criteria for PCL from outcomes of patients at mayo clinic, our patient was diagnosed with plasma cell leukemia. Given aggressive biology of this disease, he was started on VD-PACE chemotherapy. Bone marrow biopsy after cycle 1 chemotherapy showed no morphologic, immunophenotypic or flow cytometric features of a plasma cell neoplasm. Given excellent treatment response and discussion with transplant center subsequent cycle 2 was changed to Velcade, Revlimid and low-dose dexamethasone. He is scheduled for stem cell transplant later this month. Primary plasma cell leukemia (pPCL) is the most aggressive form of the plasma cell dyscrasias. The outcome of pPCL has improved with the introduction of autologous stem cell transplantation and combination approaches with novel agents, including bortezomib and immunomodulatory drugs, such as lenalidomide. This case highlights the challenges in diagnosis of non-producer primary plasma cell leukemia.

Adhesion signaling is a field of cell biology studied mostly on adherent cell types. However, hematopoietic cells grow in suspension, and use adhesion to the extracellular matrix (ECM) only in their early development, or - in case of differentiated cells - to perform the tasks they are specialized for. Peripheral leukemic cells are derived from more or less immature hematopoietic precursors that have, among other alterations, defects in adhesion to the bone marrow microenvironment. On the other hand, leukemic stem cells (LSC) use adhesion to the bone marrow ECM as a mean to evade chemotherapy, and are a source of the minimal residual disease, and of the disease relapses. Kinases of the Src family (SFK) are known regulators of adhesion signaling in adherent cell types, and their overexpression and/or hyperactivation is often seen in malignant diseases. They are also involved in hematooncologic disease progression and resistance to therapy, particularly in several types of leukemias. In the present work, we used a variety of methods including microimpedance measurement, fluorimetric measurement of adhered cell fraction, immunoblotting, confocal microscopy, and interference reflection microscopy. Our results indicate that active Lyn kinase, a hematopoietic SFK, is present in adhesion structures of...

Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...

Therapy of acute leukemia may be improved by a more accurate assessment of the effects of treatment on tumor burden and by anticipating relapse with greater precision. The sensitivity limit of assessing residual disease by morphology is usually 5%. Several alternative approaches are available to study minimal residual disease, defined as the presence of leukemic cells not detectable by morphology. These include studies of chromosomal abnormalities by conventional karyotyping, flow cytometry, in situ hybridization and polymerase chain reaction (PCR), investigation of gene rearrangements by Southern blotting and PCR, and immunological methods. Some of these techniques enable the detection of 1 leukemic cells among 10 000 or more normal cells. In the following, the advantages and limitations of sensitive methods for detecting small numbers of leukemic cells are reviewed. The rationale for monitoring residual disease in acute leukemia and the initial results of studies correlating minimal residual disease and clinical outcome are discussed.