134

Advanced Clinical Research Center

Division of Hematopoietic Disease Control
造血病態制御学分野

Professor
Yasuhito Nannya, M.D., Ph.D.
Associate Professor Takaaki Konuma, M.D., Ph.D.
Assistant Professor Seiko Kato, M.D., Ph.D.

教 授
准教授
助 教

博士（医学）
博士（医学）
博士（医学）南小加

谷沼
藤泰 仁貴 晶せい子

The main goal of our research is to elucidate the pathogenesis of hematopoietic
diseases and to study the development of therapeutic methods for these diseases.
For those therapeutic targets that have already been identified, we will continue todevelop them and move them to the next stage of clinical application. In particular,
in terms of elucidating the pathophysiology through genome analysis, we arecollaborating with various research groups in Japan and overseas, and have madeinteresting findings on myeloproliferative tumors and the clonal evolution of PIGA.
We are also leading a whole-genome sequencing project of more than 1,400
leukemia samples collected from major hematopoietic disease centers in Japan,
and will begin analyzing genomic data from these samples this fiscal year. The
project aims to comprehensively search for abnormalities that occur in non-genetic
coding regions and other regions of the genome, and to elucidate previously
undiscovered pathomechanisms. We also aim to identify the genetic predisposition
to leukemia.

1. Clonal progression mechanisms of Myeloproliferative neoplasms using whole genome sequencing across disease stages
Takamori, H1, Lee-Yung Shih3, Ying-Jung Huang3,
Ogawa S4, Nannya Y1,2
1 Division of Hematopoietic Disease Control, IMSUT,
2 Department of Hematology/Oncology, IMSUT
Hospital
3 Division of Hematology-Oncology, Department ofInternal Medicine, Chang Gung Memorial Hospitalat Linkou, Taoyuan 333423, Taiwan
4 Department of Pathology and Tumor Biology,
Kyoto University
Myeloproliferative tumors have a chronic course
in true polycythemia vera and essential thrombocythemia, some of which may progress to myelofibrosis and acute myelogenous leukemia. The genomic

aberrant changes that occur in such cases remain to beelucidated. We performed whole-genome sequencingof bone marrow fluid samples from 23 patients withmyeloproliferative tumors that had converted fromchronic to acute myelogenous leukemia before andafter conversion. Almost all of the patients had received JAK inhibitors. The results showed that inmost cases, driver mutations found in myeloid tumors appeared as subclones of mutations characteristicof MPNs. The mutation signature showed the same
pattern between the chronic phase and acute transformation, suggesting that the mutagenicity was identical for both phases. Interestingly, there were two cases of JAK2 mutation-positive MPNs that progressed
to JAK2-negative secondary AML, both with a common parental clone, a result contrary to the conventional hypothesis of secondary AML emerging fromcompletely different clones.

135
2. Insights from Trajectories of PIGA-mutatedclones in Paroxysmal Nocturnal Hemoglobinuria
Hiroyuki Takamori1),2), Yasutaka Ueda1), Yoshikazu Matsuoka3), Tatsuya Fujioka3), Hideki Makishima4), Yoshiko Murakami5), Taroh Kinoshita5),6),7),
Satoru Miyano8), Yuzuru Kanakura1),9), Jun-Ichi
Nishimura1), Naoki Hosen1), Seishi Ogawa4), Yasuhito Nannya2),4)
1)Department of Hematology and Oncology, Osaka
University Graduate School of Medicine
2)Division of Hematopoietic Disease Control, TheInstitute of Medical Science, The University of Tokyo3)Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University4)Department of Pathology and Tumor Biology,Kyoto University5)Laboratory of Immunoglycobiology, Research Institute for Microbial Diseases, Osaka University6)Immunoglycobiology, WPI Immunology Frontier
Research Center, Osaka University7)Center for Infectious Disease Education and Research, Osaka University8)M&D Data Science Center, Tokyo Medical andDental University9)Department of Hematology Sumitomo Hospital
The pathophysiology of clonal expansion withparoxysmal nocturnal hemoglobinuria (PNH) remains unclear. To elucidate the trajectories of PNHclones, we constructed phylogenetic trees using
whole genome sequencing data of single cell-derived
colonies of bone marrow samples from two PNH patients (52 colonies for case 1 and 77 for case 2). In case
1, we identified three PIGA-mutated and one
BCOR-mutated clone with no shared common somatic variants, showing independent origin. In case 2,
one PIGA-mutated clone was detected, in which
HMGA2 mutations (Inoue et al. Blood. 2006) were accompanied in all the colonies examined. Bayesian estimation revealed that these PIGA mutations are assumed to be acquired around 14 years before theclinical PNH onset in both cases. PIGA-mutatedclones exhibited a higher mutation acquisition rate
than unmutated ones (16 vs. 11 mutations/year forcase 1 and 14 vs. 12 for case 2). We estimated the effective population size of PIGA-mutated clones using
coalesce principles. PIGA-mutated clones grew more
slowly than unmutated normal clones of early embryonic development. In case 1, the onset of the significant clonal expansion of two PIGA-mutated clones
seems to coincide with diagnosis of the aplastic anemia (AA), which supports the theory that immune
attack associated with AA facilitated the expansion ofPNH clones. This technique helps to infer the diseasedevelopment mechanism by depicting the diseased
clone’s evolutionary history, which cannot be directly

observed.
3. A forward-looking analytical study of minimal
residual disease after allogeneic hematopoietic stem cell transplantation using circulatingtumor DNA in acute myeloid leukemia:
KSGCT1702
Ogawa M1, Yokoyama K2, Ishii H3, Nannya Y12,
Kanto Study & Group for Cell Therapy (KSGCT)​​
1 Division of Hematopoietic Disease Control, IMSUT2 Department of Hematology/Oncology, IMSUT
Hospital
3 Division of Clinical Oncology and Hematology,
Department of InternalMedicine, Jikei University School of Medicine
Post-allogeneic relapse of AML/MDS is an important clinical issue, and there is a need to develop a
non-invasive method to detect relapse at an earlystage. To evaluate the usefulness of the minimal residual disease assay, which measures driver mutations identified by comprehensive genetic analysis in
post-transplant AML/MDS patients by VAF of circulating tumor DNA in post-transplant patients’ serum,in transplant patients in the Kanto Hematopoietic
Stem Cell Group.
Patients diagnosed as AML/MDS cases according
to WHO Classification 2008, aged 20 to 65 years, whomay undergo allogeneic hematopoietic stem celltransplantation with myeloablative pretreatment at
KSGCT participating centers, and whose written consent has been obtained from the patient. Target cases
will be enrolled and next-generation sequencing will
be performed on tumor and control (oral mucosa)
specimens. Driver gene mutations will be identified
and a Droplet Digital PCR (ddPCR) assay will be designed. Cell free DNA will be extracted from serum
samples before and after bone marrow transplantation, and driver gene mutations will be quantitatively
measured using ddPCR. 70 cases were enrolled from
12 centers, of which 12 cases dropped out before
transplantation. Transplantation was performed in 58
cases, recurrence within 1 year after transplantationin 15 cases, and death within 1 year after transplantation in 10 cases (preimplantation death in 1 case, recurrence death in 4 cases). Of the transplant recipients, 57 underwent NGS to identify driver mutations.
52 were identified as MRD-eligible driver mutations.
48 completed MRD measurement by ddPCR.
4. A forward-looking analytical study of minimal
residual disease after allogeneic hematopoietic stem cell transplantation using circulatingtumor DNA in acute lymphoid leukemia:
KSGCT1901
Ogawa M1, Yokoyama K2, Ishii H3, Nannya Y12,

136
Kanto Study & Group for Cell Therapy (KSGCT)​​
1 Division of Hematopoietic Disease Control, IMSUT
2 Department of Hematology/Oncology, IMSUT
Hospital
3 Division of Clinical Oncology and Hematology,
Department of Internal
Post-allogeneic relapse of ALL is an important
clinical issue, and there is a need to develop a non-invasive method to detect relapse at an early stage. To
evaluate the usefulness of the minimal residual disease assay, which measures driver mutations identified by comprehensive genetic analysis in post-transplant ALL patients by VAF of circulating tumor DNA
in post-transplant patients’ serum, in transplant patients in the Kanto Hematopoietic Stem Cell Group.
Patients 16 years of age or older, acute lymphoblastic leukemia according to WHO classification
2016, any history of chemotherapy at the time oftransplantation, specimens with tumor volume of atleast 20% available, potential for allogeneic transplantation, and written consent obtained from the patient.
Target cases will be enrolled and next-generation sequencing will be performed on tumor and control(oral mucosa) specimens. Driver gene mutations willbe identified and a Droplet Digital PCR (ddPCR) assay will be designed. Cell free DNA will be extracted
from serum samples before and after bone marrowtransplantation, and driver gene mutations will bequantitatively measured using ddPCR. There were 54
cases enrolled, 5 cases of pre-transplant dropout, and43 cases of transplantation performed. NGS of tumor
samples was performed in 40 cases and driver genemutations were identified in 24 cases. A Droplet Digital PCR assay is being designed for each case.
5. Novel recurrent structural variants in hematopoietic malignancies and construction of Japanese-SV-specific panel of normal (PoN)
Fukushima H1,2, Ogawa S3, Nannya Y1,2
1 Division of Hematopoietic Disease Control, IMSUT,
2 Department of Hematology/Oncology, IMSUT
Hospital
3 Department of Pathology and Tumor Biology,
Kyoto University
Structural Variation analysis was performed withGRIDSS/MANTA/GenomonSV on a total of 1453 hematological paired whole genome sequencing(WGS)
data. We found that the intersection of calls fromGRIDSS, MANTA, GenomonSV, Delly and SVABA
was less than 1%, and the vast majority of them arepresumed to be false positive calls. This is mainly due
to the inability of every caller to remove alignment
errors at repetitive or low-complexity sequences. We,
therefore, constructed Japanese specific Structural
Variant Panel of Normal (PoN) with recurrent callsfrom samples of healthy japanese donors, aiming toprovide a set of false-positive calls that should be subtracted from the call results of Japanese tumour samples. We confirmed a significant (>80%) reduction in
the number of calls using this panel, and currently
figuring out novel recurrent pathogenic structuralvariants.

Publications1.		Abe S, Tago S, Yokoyama K, Ogawa M, Takei T,
Imoto S, Fuji M. Explainable AI for Estimating
Pathogenicity of Genetic Variants Using LargeScale Knowledge Graphs. Cancers (Basel).2023,15(4),2.		Edahiro R, Shirai Y, Takeshima Y, Sakakibara S,Yamaguchi Y, Murakami T, Morita T, Kato Y, LiuYC, Motooka D, Naito Y, Takuwa A, Sugihara F,Tanaka K, Wing JB, Sonehara K, Tomofuji Y, Japan
C-TF, Namkoong H, Tanaka H, Lee H, Fukunaga
K, Hirata H, Takeda Y, Okuzaki D, Kumanogoh A,Okada Y. Single-cell analyses and host genetics
highlight the role of innate immune cells in COVID-19 severity. Nat Genet.2023, 55(5),753-67.
3.		Elbadry MI, Tawfeek A, Hirano T, El-Mokhtar
MA, Kenawey M, Helmy AM, Ogawa S, Mughal
MZ, Nannya Y. A rare homozygous variant in
TERT gene causing variable bone marrow failure,fragility fractures, rib anomalies and extremely
short telomere lengths with high serum IgE. Br J
Haematol.2023,

4.		Initiative C-HG. A second update on mapping thehuman genetic architecture of COVID-19. Nature.2023, 621(7977),E7-E26.
5.		Jimbo K, Hattori A, Koide S, Ito T, Sasaki K, IwaiK, Nannya Y, Iwama A, Tojo A, Konuma T. Genetic deletion and pharmacologic inhibition of E3
ubiquitin ligase HOIP impairs the propagation ofmyeloid leukemia. Leukemia.2023, 37(1),122-33.
6.		Kubota H, Ueno H, Tasaka K, Isobe T, Saida S,Kato I, Umeda K, Hiwatari M, Hasegawa D, Imamura T, Kakiuchi N, Nannya Y, Ogawa S, Hiramatsu H, Takita J. RNA-seq-based miRNA signature as an independent predictor of relapse inpediatric B-cell acute lymphoblastic leukemia.
Blood Adv.2023,7.		Makishima H, Saiki R, Nannya Y, Korotev S, Gurnari C, Takeda J, Momozawa Y, Best S, Krishnamurthy P, Yoshizato T, Atsuta Y, Shiozawa Y, Iijima-Yamashita Y, Yoshida K, Shiraishi Y, Nagata Y,Kakiuchi N, Onizuka M, Chiba K, Tanaka H, Kon
A, Ochi Y, Nakagawa MM, Okuda R, Mori T, Yoda

137
A, Itonaga H, Miyazaki Y, Sanada M, Ishikawa T,Chiba S, Tsurumi H, Kasahara S, Muller-Tidow C,Takaori-Kondo A, Ohyashiki K, Kiguchi T, Matsuda F, Jansen JH, Polprasert C, Blombery P,Kamatani Y, Miyano S, Malcovati L, Haferlach T,Kubo M, Cazzola M, Kulasekararaj AG, Godley
LA, Maciejewski JP, Ogawa S. Germ line DDX41
mutations define a unique subtype of myeloid neoplasms. Blood.2023, 141(5),534-49.
8.		Nannya Y, Tobiasson M, Sato S, Bernard E, Ohtake
S, Takeda J, Creignou M, Zhao L, Kusakabe M,Shibata Y, Nakamura N, Watanabe M, Hiramoto
N, Shiozawa Y, Shiraishi Y, Tanaka H, Yoshida K,Kakiuchi N, Makishima H, Nakagawa M, Usuki
K, Watanabe M, Imada K, Handa H, Taguchi M,Kiguchi T, Ohyashiki K, Ishikawa T, Takaori-Kondo A, Tsurumi H, Kasahara S, Chiba S, Naoe T,Miyano S, Papaemanuil E, Miyazaki Y, Hellstrom-Lindberg E, Ogawa S. Postazacitidine clone
size predicts long-term outcome of patients withmyelodysplastic syndromes and related myeloid

neoplasms. Blood Adv.2023, 7(14),3624-36.
9.		Nishimura T, Kakiuchi N, Yoshida K, Sakurai T,
Kataoka TR, Kondoh E, Chigusa Y, Kawai M,Sawada M, Inoue T, Takeuchi Y, Maeda H, Baba S,Shiozawa Y, Saiki R, Nakagawa MM, Nannya Y,Ochi Y, Hirano T, Nakagawa T, Inagaki-Kawata Y,
Aoki K, Hirata M, Nanki K, Matano M, Saito M,Suzuki E, Takada M, Kawashima M, Kawaguchi
K, Chiba K, Shiraishi Y, Takita J, Miyano S, Mandai
M, Sato T, Takeuchi K, Haga H, Toi M, Ogawa S.
Evolutionary histories of breast cancer and relatedclones. Nature.2023, 620(7974),607-14.
10.	Yabushita T, Chinen T, Nishiyama A, Asada S, Shimura R, Isobe T, Yamamoto K, Sato N, Enomoto Y,Tanaka Y, Fukuyama T, Satoh H, Kato K, Saitoh K,Ishikawa T, Soga T, Nannya Y, Fukagawa T, Nakanishi M, Kitagawa D, Kitamura T, Goyama S. Mitotic perturbation is a key mechanism of action ofdecitabine in myeloid tumor treatment. CellRep.2023, 42(9),113098.