Faculty Profiles - ENNISHI Daisuke

Papers - ENNISHI Daisuke

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Single Cell Phenotypic Profiling of 27 DLBCL Cases Reveals Marked Intertumoral and Intratumoral Heterogeneity. International journal

Michael D Nissen, Manabu Kusakabe, Xuehai Wang, Guillermo Simkin, Deanne Gracias, Kateryna Tyshchenko, Ainsleigh Hill, Justin Meskas, Stacy Hung, Elizabeth A Chavez, Daisuke Ennishi, Tomohiro Aoki, Clementine Sarkozy, Joseph M Connors, Pedro Farinha, Graham W Slack, Randy D Gascoyne, Ryan R Brinkman, David W Scott, Christian Steidl, Andrew P Weng

Cytometry. Part A : the journal of the International Society for Analytical Cytology 97 ( 6 ) 620 - 629 2020.6

Language：English Publishing type：Research paper (scientific journal)

Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry.

DOI： 10.1002/cyto.a.23919

An Endoscopic Biopsy Specimen Contains Adequate Lymphocytes for Flow Cytometric Analysis of Light Chain Expression in the Gastrointestinal Mucosa. International journal

Masaya Iwamuro, Katsunori Matsueda, Takahide Takahashi, Sizuma Omote, Takehiro Tanaka, Daisuke Ennishi, Fumio Otsuka, Tadashi Yoshino, Hiroyuki Okada

Annals of clinical and laboratory science 50 ( 3 ) 348 - 353 2020.5

Language：English Publishing type：Research paper (scientific journal)

OBJECTIVE: Flow cytometry has not been widely used in routine clinical practice for the diagnosis of gastrointestinal lymphoma; this is mainly because of the absence of an appropriate protocol. Here, we established a protocol for flow cytometric analysis of a single biopsy specimen from the gastrointestinal mucosa and investigated its sensitivity and specificity. DESIGN: In this prospective study, we enrolled patients with previously diagnosed gastrointestinal lymphoma and patients with gastrointestinal lesions that were suspected to be lymphoma. RESULTS: Overall, 15 patients with gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (N=8), duodenal follicular lymphoma (grade 1; N=5), and benign lymphoid hyperplasia (ileum, N=1, and rectum, N=1) were included in this study. Of these, lymphocytes were isolated from 14 patients (93.3%). There were 200,000-1,500,000 viable cells per patient. Biopsy specimens from 10 out of the 12 patients with lymphoma were positive for light chain restriction; the two patients with benign lymphoid hyperplasia showed negative results. CONCLUSIONS: An adequate number of lymphocytes for flow cytometry could be isolated from a single specimen of endoscopic mucosal biopsy from 93.3% of the patients. Overall, the sensitivity of flow cytometric analysis of light chain expression for the diagnosis of B-cell lymphoma was 83.3%, and the specificity was 100%. Although further investigation is required as the sample size of the present study was small, our study suggests a potential option for diagnosing B-cell lymphoma in the gastrointestinal mucosa.

Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone combined with high-dose methotrexate plus intrathecal chemotherapy for newly diagnosed intravascular large B-cell lymphoma (PRIMEUR-IVL): a multicentre, single-arm, phase 2 trial. International journal

Kazuyuki Shimada, Motoko Yamaguchi, Yoshiko Atsuta, Kosei Matsue, Keijiro Sato, Shigeru Kusumoto, Hirokazu Nagai, Jun Takizawa, Noriko Fukuhara, Koji Nagafuji, Kana Miyazaki, Eiichi Ohtsuka, Masataka Okamoto, Yasumasa Sugita, Toshiki Uchida, Satoshi Kayukawa, Atsushi Wake, Daisuke Ennishi, Yukio Kondo, Tohru Izumi, Yoshihiro Kin, Kunihiro Tsukasaki, Daigo Hashimoto, Masaaki Yuge, Atsumi Yanagisawa, Yachiyo Kuwatsuka, Satoko Shimada, Yasufumi Masaki, Nozomi Niitsu, Hitoshi Kiyoi, Ritsuro Suzuki, Takashi Tokunaga, Shigeo Nakamura, Tomohiro Kinoshita

The Lancet. Oncology 21 ( 4 ) 593 - 602 2020.4

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Intravascular large B-cell lymphoma (IVLBCL) is a rare disease for which there is no available standard treatment. We aimed to ascertain the safety and activity of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) with high-dose methotrexate and intrathecal chemotherapy as CNS-oriented therapy for patients with previously untreated IVLBCL. METHODS: PRIMEUR-IVL is a multicentre, single-arm, phase 2 trial at 22 hospitals in Japan. Eligible patients had untreated histologically confirmed IVLBCL, were aged 20-79 years, had an Eastern Cooperative Group performance status of 0-3, and had no apparent CNS involvement at diagnosis. Patients received three cycles of R-CHOP (rituximab 375 mg/m2 intravenously on day 1 [except cycle one, which was on day 8]; cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1·4 mg/m2 [maximum 2·0 mg] intravenously on day 1 of cycle one and day 2 of cycles two and three; and prednisolone 100 mg/day orally on days 1-5 of cycle one and days 2-6 of cycles two and three) followed by two cycles of rituximab with high-dose methotrexate (3·5 g/m2 intravenously on day 2 of cycles four and five) every 2 weeks and three additional cycles of R-CHOP. Intrathecal chemotherapy (methotrexate 15 mg, cytarabine 40 mg, and prednisolone 10 mg) was administered four times during the R-CHOP phase. The primary endpoint was 2-year progression-free survival. Efficacy analyses were done in all enrolled patients; safety analyses were done in all enrolled and treated patients. The trial is registered in the UMIN Clinical Trials Registry (UMIN000005707) and the Japan Registry of Clinical Trials (jRCTs041180165); the trial is ongoing for long-term follow-up. FINDINGS: Between June 16, 2011, and July 21, 2016, 38 patients were enrolled, of whom 37 were eligible; one patient was excluded because of a history of testicular lymphoma. Median follow-up was 3·9 years (IQR 2·5-5·5). 2-year progression-free survival was 76% (95% CI 58-87). The most frequent adverse events of grade 3-4 were neutropenia and leucocytopenia, which were reported in all 38 (100%) patients. Serious adverse events were hypokalaemia, febrile neutropenia with hypotension, hypertension, and intracerebral haemorrhage (reported in one [3%] patient each). No treatment-related deaths occurred during protocol treatment. INTERPRETATION: R-CHOP combined with rituximab and high-dose methotrexate plus intrathecal chemotherapy is a safe and active treatment for patients with IVLBCL without apparent CNS involvement at diagnosis, and this regimen warrants future investigation. FUNDING: The Japan Agency for Medical Research and Development, the Center for Supporting Hematology-Oncology Trials, and the National Cancer Center.

DOI： 10.1016/S1470-2045(20)30059-0

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma. International journal

Daisuke Ennishi, Shannon Healy, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Libin Abraham, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W Woolcock, Elena Viganò, Michael Gold, Laurie L Molday, Robert S Molday, Adele Telenius, Michael Y Li, Nicole Wretham, Nancy Dos Santos, Mark Wong, Natasja N Viller, Robert A Uger, Gerben Duns, Abigail Baticados, Angel Madero, Brianna N Bristow, Pedro Farinha, Graham W Slack, Susana Ben-Neriah, Daniel Lai, Allen W Zhang, Sohrab Salehi, Hennady P Shulha, Derek S Chiu, Sara Mostafavi, Alina S Gerrie, Da Wei Huang, Christopher Rushton, Diego Villa, Laurie H Sehn, Kerry J Savage, Andrew J Mungall, Andrew P Weng, Marcel B Bally, Ryan D Morin, Gabriela V Cohen Freue, Louis M Staudt, Joseph M Connors, Marco A Marra, Sohrab P Shah, Randy D Gascoyne, David W Scott, Christian Steidl

Nature medicine 26 ( 4 ) 577 - 588 2020.4

Language：English Publishing type：Research paper (scientific journal)

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

DOI： 10.1038/s41591-020-0757-z

Efficacy of HLA virtual cross-matched platelet transfusions for platelet transfusion refractoriness in hematopoietic stem cell transplantation. International journal

Keisuke Seike, Nobuharu Fujii, Naomi Asano, Shigenori Ohkuma, Yasushi Hirata, Keiko Fujii, Yasuhisa Sando, Makoto Nakamura, Kazunori Naito, Kyosuke Saeki, Yusuke Meguri, Noboru Asada, Daisuke Ennishi, Hisakazu Nishimori, Ken-Ichi Matsuoka, Kazuo Tsubaki, Fumio Otsuka, Yoshinobu Maeda

Transfusion 60 ( 3 ) 473 - 478 2020.3

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Cross-matched platelet (cross-matched PLT) transfusion is effective for immune-mediated platelet transfusion refractoriness (PTR), but is more costly and time-consuming for physical cross-match than using standard PLT units. Recent studies have reported the utility of human leucocyte antigens (HLA) virtual cross-matched PLT (HLA-matched PLT) that is defined as HLA-A/B matched or no antibody against donor-specific antigen. Here, we evaluated the effect of HLA-matched PLTs for PTR in post hematopoietic stem cell transplant (HSCT) recipients. STUDY DESIGN AND METHODS: Our study included a total of 241 PLTs in 16 patients who underwent HSCT at Okayama University Hospital between 2010 and 2017, receiving either HLA-matched or cross-matched PLTs. We calculated the 24-hour corrected count increments (CCI-24) to evaluate the effect of PLTs. A CCI-24 ≥ 4500 was considered to be a successful transfusion. RESULTS: We analyzed 139 cross-matched PLTs and 102 HLA-matched PLTs. In the immune-mediated PTR, the rate of successful transfusion was 60.5% for cross-matched PLT and 63.4% for HLA-matched PLT (p = 0.825). On the other hand, the median CCI-24 for cross-matched PLT transfusions and HLA-matched PLT transfusions were 1856 and 5824 (p < 0.001), with a success rate of 28.1 and 54.1% in cases with non-immune-mediated PTR, respectively (p = 0.001). CONCLUSION: The effectiveness of HLA-matched PLT is not inferior to cross-matched PLT. This result indicates that physical cross-match can be omitted in post HSCT PTR.

DOI： 10.1111/trf.15664

Technique for single-step lymphocyte isolation from an endoscopic biopsy specimen for the diagnosis of gastrointestinal lymphoma. International journal

Masaya Iwamuro, Takahide Takahashi, Natsuki Watanabe, Sizuma Omote, Katsunori Matsueda, Takehiro Tanaka, Daisuke Ennishi, Fumio Otsuka, Tadashi Yoshino, Hiroyuki Okada

MethodsX 7 101095 - 101095 2020

Language：English Publishing type：Research paper (scientific journal)

In this paper, we introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment. For lymphocyte isolation, an endoscopically harvested specimen and 5 mL of normal saline solution were placed in a wire mesh strainer set in a porcelain bowl. To obtain the lymphocyte suspension, the solid specimen was crushed using the rubber portion of a plunger of a 10 mL injection syringe. Flow cytometry was performed using the lymphocyte suspension. For validating our methods, the one-step lymphocyte isolation technique was used to perform flow cytometry on samples from 23 patients with (n = 12) or without (n = 11) gastrointestinal lymphoma. Flow cytometry of light chain expression was performed in all patient samples (feasibility: 100%). Sensitivity was 83.3% (10/12) and specificity was 100% (11/11). In conclusion, lymphocytes isolated from a single endoscopic biopsy specimen using our simplified and quick procedure are suitable for flow cytometry. Considering that flow cytometry has an important advantage of providing the results on the examination day itself, the results of this study suggest that flow cytometric analysis using our single-step lymphocyte isolation technique can be potentially used to diagnose lymphoma in the gastrointestinal mucosa. •We introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment.•Our technique is feasible for flow cytometric analysis in patients with gastrointestinal lymphoma as well as those with gastrointestinal lesions that are suspected to be lymphoma.

DOI： 10.1016/j.mex.2020.101095

Diffuse Large B-Cell Lymphomas with a Molecular PMBCL Expression Signature Represent a Distinct Molecular Subtype Associated with Poor Clinical Outcome

Gerben Duns, Elena Vigano, Daisuke Ennishi, Clementine Sarkozy, Stacy Hung, Elizabeth Chavez, Katsuyoshi Takata, Anja Mottok, Randy D. Gascoyne, Ryan D. Morin, Kerry J. Savage, David W. Scott, Christian Steidl

BLOOD 134 2019.11

Feasibility of flow cytometric analysis of restricted light chain in endoscopic biopsy specimens from patients with gastrointestinal tract B cell lymphoma: a pilot study. International journal

Katsunori Matsueda, Masaya Iwamuro, Takahide Takahashi, Sizuma Omote, Kenji Nishida, Takehiro Tanaka, Daisuke Ennishi, Fumio Otsuka, Tadashi Yoshino, Hiroyuki Okada

BMC research notes 12 ( 1 ) 571 - 571 2019.9

Language：English Publishing type：Research paper (scientific journal)

OBJECTIVE: Gastrointestinal tract lymphomas are currently detected more frequently due to advances in endoscopic technology. The aim of this study was to assess the feasibility of flow cytometric analysis of restricted light chain in endoscopic biopsy specimens for the diagnosis of gastrointestinal tract B-cell lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 10 consecutive patients who had a previous histological diagnosis of gastrointestinal tract B-cell lymphoma. We performed immunophenotypic studies with multi-color flow cytometry and assessed clonality through examination of immunoglobulin light chain expression exclusively in a population identified by anti-CD45 or CD20 antibodies. RESULTS: We could perform light chain expression analysis with 2 endoscopic biopsy specimens from all 10 patients with gastrointestinal tract B-cell lymphoma. We conclude that flow cytometric analysis of endoscopic biopsy specimens is feasible and thus likely useful for the diagnosis of gastrointestinal tract B-cell lymphoma in clinical settings. Trial registration UMIN Clinical Trials Registry, UMIN000027730. Registered 12 June 2017.

DOI： 10.1186/s13104-019-4578-4

Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 as Therapeutic Target for Enhancing Immune Recognition. International journal

Daisuke Ennishi, Katsuyoshi Takata, Wendy Béguelin, Gerben Duns, Anja Mottok, Pedro Farinha, Ali Bashashati, Saeed Saberi, Merrill Boyle, Barbara Meissner, Susana Ben-Neriah, Bruce W Woolcock, Adèle Telenius, Daniel Lai, Matt Teater, Robert Kridel, Kerry J Savage, Laurie H Sehn, Ryan D Morin, Marco A Marra, Sohrab P Shah, Joseph M Connors, Randy D Gascoyne, David W Scott, Ari M Melnick, Christian Steidl

Cancer discovery 9 ( 4 ) 546 - 563 2019.4

Language：English Publishing type：Research paper (scientific journal)

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II-deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of EZH2 mutations in both MHC-I- and MHC-II-negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant Ezh2 Y641. Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in EZH2-mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming. SIGNIFICANCE: We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin-specific context. Specifically, EZH2 mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination.See related commentary by Velcheti et al., p. 472.This article is highlighted in the In This Issue feature, p. 453.

DOI： 10.1158/2159-8290.CD-18-1090

Salvage Haploidentical Transplantation Using Low-dose ATG for Early Disease Relapse after First Allogeneic Transplantation: A Retrospective Single-center Review.

Sachiyo Okamoto, Ken-Ichi Matsuoka, Maiko Sakamoto, Yoshiaki Usui, Yuki Fujiwara, Takumi Kondo, Katsuma Tani, Kyosuke Saeki, Yusuke Meguri, Noboru Asada, Daisuke Ennishi, Hisakazu Nishimori, Keiko Fujii, Nobuharu Fujii, Yoshinobu Maeda

Acta medica Okayama 73 ( 2 ) 161 - 171 2019.4

Language：English Publishing type：Research paper (scientific journal)

Second allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for patients who relapse after first allo-SCT. Human leukocyte antigen (HLA)-haploidentical related donors provide the broad opportunity to conduct second SCT at the appropriate time, but the efficacy of second SCT from haploidentical donors after relapse has not been established. We retrospectively analyzed the records of 33 patients who underwent second SCT. Twenty patients underwent haplo-SCT with low-dose antithymocyte globulin (ATG), and the other 13 patients underwent conventional- SCTs, including HLA-matched related peripheral blood, unrelated bone marrow or cord blood. Three years after the second SCT, the overall survival (OS) and progression-free survival (PFS) of all patients were 32.5% and 23.9%. Multivariate analyses indicated that non-complete response at second SCT, less than 1-year interval to relapse after first- SCT, and total score ≥ 3 on the hematopoietic cell transplantation-specific comorbidity index were significantly associated with a lower PFS rate. The haplo- and conventional- SCT groups showed equivalent results regarding OS, PFS, cumulative incidences of relapse, non-relapse mortality and graft-versus-host disease. The neutropenic period after transplantation was significantly shorter in haplo- SCT than conventional- SCT (10.5 days vs. 16 days, p=0.001). Our analysis revealed that haplo-SCT could be an alternative therapeutic option for relapsed patients after first SCT.

DOI： 10.18926/AMO/56652

Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. International journal

Daisuke Ennishi, Aixiang Jiang, Merrill Boyle, Brett Collinge, Bruno M Grande, Susana Ben-Neriah, Christopher Rushton, Jeffrey Tang, Nicole Thomas, Graham W Slack, Pedro Farinha, Katsuyoshi Takata, Tomoko Miyata-Takata, Jeffrey Craig, Anja Mottok, Barbara Meissner, Saeed Saberi, Ali Bashashati, Diego Villa, Kerry J Savage, Laurie H Sehn, Robert Kridel, Andrew J Mungall, Marco A Marra, Sohrab P Shah, Christian Steidl, Joseph M Connors, Randy D Gascoyne, Ryan D Morin, David W Scott

Journal of clinical oncology : official journal of the American Society of Clinical Oncology 37 ( 3 ) 190 - 201 2019.1

Language：English Publishing type：Research paper (scientific journal)

PURPOSE: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression. PATIENTS AND METHODS: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data. RESULTS: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested. CONCLUSION: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.

DOI： 10.1200/JCO.18.01583

[B-cell lymphomas].

Daisuke Ennishi

[Rinsho ketsueki] The Japanese journal of clinical hematology 60 ( 5 ) 434 - 440 2019

Language：Japanese Publisher：The Japanese Society of Hematology

Among the hematologic tumors, lymphoid malignancies, especially B-cell lymphomas (BLs), are categorized according to the WHO classification into a large number of entities, which are aggregates of clinically and biologically diverse diseases. It is thought that various molecular genetic abnormalities are involved in lymphoid tumorigenesis. Recent advances in genetic analysis technology have resulted in the discovery of many novel and recurrent genetic abnormalities. In particular, those associated with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma have been identified utilizing molecular pathology techniques, which is important aspect for the future development of novel therapeutic drugs and indications for clinical trials. In this section, the possibility of patient-stratification based on genetic abnormalities of DLBCL and FL, which are particularly prevalent among mature BLs, are described. I also introduce the genetic features and clinical importance of BL and MCL identified in a small number of patients in Japan.

DOI： 10.11406/rinketsu.60.434

Other Link： http://search.jamas.or.jp/link/ui/2019327541

[Molecular classification and its clinical relevance in diffuse large B-cell lymphoma].

Daisuke Ennishi

[Rinsho ketsueki] The Japanese journal of clinical hematology 60 ( 9 ) 1186 - 1192 2019

Language：Japanese Publisher：The Japanese Society of Hematology

Hematopoietic tumors represent a range of clinically and biologically diverse diseases, and within this, the lymphoid malignancies, especially B-cell lymphomas, are classified into many entities in the WHO classification system. It is thought that various molecular genetic abnormalities are involved in the tumorigenesis of diffuse large B-cell lymphoma (DLBCL). Due to recent developments in genetic analysis technology, novel and recurrent genetic abnormalities have been discovered. This is important knowledge for the development of new therapeutic drugs and may support indications for clinical trials, ultimately leading to the availability of more targeted therapies and precision medicine in the DLBCL arena. Here the possibility of patient stratification, based on genetic abnormality, has been described.

DOI： 10.11406/rinketsu.60.1186

Other Link： https://search.jamas.or.jp/link/ui/2020081718

High-resolution architecture and partner genes of MYC rearrangements in lymphoma with DLBCL morphology. International journal

Lauren C Chong, Susana Ben-Neriah, Graham W Slack, Ciara Freeman, Daisuke Ennishi, Anja Mottok, Brett Collinge, Pau Abrisqueta, Pedro Farinha, Merrill Boyle, Barbara Meissner, Robert Kridel, Alina S Gerrie, Diego Villa, Kerry J Savage, Laurie H Sehn, Reiner Siebert, Ryan D Morin, Randy D Gascoyne, Marco A Marra, Joseph M Connors, Andrew J Mungall, Christian Steidl, David W Scott

Blood advances 2 ( 20 ) 2755 - 2765 2018.10

Language：English Publishing type：Research paper (scientific journal)

Genomic rearrangements in the MYC locus occur in ∼12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring MYC rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of MYC, BCL2, BCL6, and the immunoglobulin (IG) loci in 112 tumors with DLBCL morphology harboring MYC rearrangement. We characterized the location of the MYC rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the MYC coding region and in intron 1 (the "genic cluster"). Genic cluster rearrangements were enriched for translocations involving IGH (80%), whereas nongenic rearrangements occurred mostly downstream of the MYC gene with a variety of partners, including IGL and IGK Other recurrent partners included BCL6, ZCCHC7, and RFTN1, which has not previously been described as a MYC partner. We compared 2 commercially available FISH break-apart assays for the MYC locus and observed discordant results in 32% of cases examined, including some with MYC-IGL and MYC-IGK rearrangements. In cases of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH), so-called "double-hit" lymphomas, the majority of MYC rearrangements had non-IG partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, MYC-IG rearrangements showed a trend toward inferior time to progression and overall survival compared with MYC-non-IG rearrangements. Our data reveal clinically relevant architecture of MYC rearrangements in lymphomas with DLBCL morphology.

DOI： 10.1182/bloodadvances.2018023572

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma. International journal

Sarah E Arthur, Aixiang Jiang, Bruno M Grande, Miguel Alcaide, Razvan Cojocaru, Christopher K Rushton, Anja Mottok, Laura K Hilton, Prince Kumar Lat, Eric Y Zhao, Luka Culibrk, Daisuke Ennishi, Selin Jessa, Lauren Chong, Nicole Thomas, Prasath Pararajalingam, Barbara Meissner, Merrill Boyle, Jordan Davidson, Kevin R Bushell, Daniel Lai, Pedro Farinha, Graham W Slack, Gregg B Morin, Sohrab Shah, Dipankar Sen, Steven J M Jones, Andrew J Mungall, Randy D Gascoyne, Timothy E Audas, Peter Unrau, Marco A Marra, Joseph M Connors, Christian Steidl, David W Scott, Ryan D Morin

Nature communications 9 ( 1 ) 4001 - 4001 2018.10

Language：English Publishing type：Research paper (scientific journal)

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.

DOI： 10.1038/s41467-018-06354-3

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements with diffuse large B-cell lymphoma morphology. International journal

David W Scott, Rebecca L King, Annette M Staiger, Susana Ben-Neriah, Aixiang Jiang, Heike Horn, Anja Mottok, Pedro Farinha, Graham W Slack, Daisuke Ennishi, Norbert Schmitz, Michael Pfreundschuh, Grzegorz S Nowakowski, Brad S Kahl, Joseph M Connors, Randy D Gascoyne, German Ott, William R Macon, Andreas Rosenwald

Blood 131 ( 18 ) 2060 - 2064 2018.5

Language：English Publishing type：Research paper (scientific journal)

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a newly defined entity in the latest World Health Organization Classification. Accurate diagnosis would appear to mandate fluorescence in situ hybridization (FISH) for all tumors with diffuse large B-cell lymphoma (DLBCL) morphology. We present the results of FISH, cell-of-origin, and immunohistochemistry (IHC) testing from 1228 DLBCL biopsies from 3 clinical trials and a population-based registry. HGBL-DH/TH made up 7.9% of the DLBCL, confined primarily to the germinal center B-cell-like (GCB; 13.3%) compared with activated B-cell-like (ABC; 1.7%) subtype (P < .001). HGBL-DH/TH with BCL2 rearrangement is a GCB phenomenon with no cases observed in 415 ABC DLBCL. A screening strategy restricting FISH testing to tumors of GCB subtype (by Lymph2Cx or Hans IHC) plus dual protein expression of MYC and BCL2 by IHC could limit testing to 11% to 14% of tumors, with a positive predictive value of 30% to 37%; however, this strategy would miss approximately one-quarter of tumors with HBGL-DH/TH with BCL2 rearrangement and one-third of all HGBL-DH/TH. These results provide accurate estimation of the proportion of HGBL-DH/TH among tumors with DLBCL morphology and allow determination of the impact of various methods available to screen DLBCL tumors for FISH testing.

DOI： 10.1182/blood-2017-12-820605

Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management. International journal

Stacy S Hung, Barbara Meissner, Elizabeth A Chavez, Susana Ben-Neriah, Daisuke Ennishi, Martin R Jones, Hennady P Shulha, Fong Chun Chan, Merrill Boyle, Robert Kridel, Randy D Gascoyne, Andrew J Mungall, Marco A Marra, David W Scott, Joseph M Connors, Christian Steidl

The Journal of molecular diagnostics : JMD 20 ( 2 ) 203 - 214 2018.3

Language：English Publishing type：Research paper (scientific journal)

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.

DOI： 10.1016/j.jmoldx.2017.11.010

AICDA drives epigenetic heterogeneity and accelerates germinal center-derived lymphomagenesis. International journal

Matt Teater, Pilar M Dominguez, David Redmond, Zhengming Chen, Daisuke Ennishi, David W Scott, Luisa Cimmino, Paola Ghione, Jayanta Chaudhuri, Randy D Gascoyne, Iannis Aifantis, Giorgio Inghirami, Olivier Elemento, Ari Melnick, Rita Shaknovich

Nature communications 9 ( 1 ) 222 - 222 2018.1

Language：English Publishing type：Research paper (scientific journal)

Epigenetic heterogeneity is emerging as a feature of tumors. In diffuse large B-cell lymphoma (DLBCL), increased cytosine methylation heterogeneity is associated with poor clinical outcome, yet the underlying mechanisms remain unclear. Activation-induced cytidine deaminase (AICDA), an enzyme that mediates affinity maturation and facilitates DNA demethylation in germinal center (GC) B cells, is required for DLBCL pathogenesis and linked to inferior outcome. Here we show that AICDA overexpression causes more aggressive disease in BCL2-driven murine lymphomas. This phenotype is associated with increased cytosine methylation heterogeneity, but not with increased AICDA-mediated somatic mutation burden. Reciprocally, the cytosine methylation heterogeneity characteristic of normal GC B cells is lost upon AICDA depletion. These observations are relevant to human patients, since DLBCLs with high AICDA expression manifest increased methylation heterogeneity vs. AICDA-low DLBCLs. Our results identify AICDA as a driver of epigenetic heterogeneity in B-cell lymphomas with potential significance for other tumors with aberrant expression of cytidine deaminases.

DOI： 10.1038/s41467-017-02595-w

Genetic profiling of MYC and BCL2 in diffuse large B-cell lymphoma determines cell-of-origin-specific clinical impact. International journal

Daisuke Ennishi, Anja Mottok, Susana Ben-Neriah, Hennady P Shulha, Pedro Farinha, Fong Chun Chan, Barbara Meissner, Merrill Boyle, Christoffer Hother, Robert Kridel, Daniel Lai, Saeed Saberi, Ali Bashashati, Sohrab P Shah, Ryan D Morin, Marco A Marra, Kerry J Savage, Laurie H Sehn, Christian Steidl, Joseph M Connors, Randy D Gascoyne, David W Scott

Blood 129 ( 20 ) 2760 - 2770 2017.5

Language：English Publishing type：Research paper (scientific journal)

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

DOI： 10.1182/blood-2016-11-747022

Clinical and prognostic significance of aberrant T-cell marker expression in 225 cases of de novo diffuse large B-cell lymphoma and 276 cases of other B-cell lymphomas. International journal

Naoko Tsuyama, Daisuke Ennishi, Masahiro Yokoyama, Satoko Baba, Reimi Asaka, Yuko Mishima, Yasuhito Terui, Kiyohiko Hatake, Kengo Takeuchi

Oncotarget 8 ( 20 ) 33487 - 33500 2017.5

Language：English Publishing type：Research paper (scientific journal)

Expression of T-cell markers, generally investigated for immunophenotyping of T-cell lymphomas, is also observed in several types of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We previously reported that CD5 expression in DLBCL is an inferior prognostic factor in the era of rituximab. However, data regarding the frequencies, histological relevance, and prognostic importance of T-cell markers other than CD5 are currently unavailable. In the present study, we comprehensively evaluated the expression of T-cell markers (CD2, CD3, CD4, CD5, CD7, and CD8) in 501 B-cell lymphomas, including 225 DLBCLs, by flow cytometry and subsequent immunohistochemistry. T-cell markers other than CD5, such as CD2, CD4, CD7, and CD8, were expressed in 27 (5%) patients, and notably, all of these cases were classified as large B-cell lymphoma subtypes: 25 DLBCLs and 2 intravascular large B-cell lymphomas. CD5 and other T-cell markers were expressed in 15% (31/225) and 10% (25/225) of DLBCL cases, respectively. Five of them co-expressed CD5 and other T-cell markers. Retrospectively analyzing the prognostic relevance of T-cell markers in 169 patients with primary DLBCL treated with rituximab-based chemotherapy, we showed that only CD5 was a strong predictor of poor survival. This study provides information about the occurrence of T-cell markers other than CD5 in B-cell lymphomas, their frequent histological subtypes, and their prognostic significance in DLBCL. CD5 was reconfirmed as a negative prognostic marker in DLBCL patients receiving rituximab-inclusive chemotherapy, whereas T-cell markers other than CD5 were found to have no impact on clinicopathological and survival analyses.

DOI： 10.18632/oncotarget.16532

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