Japan Blood Products Organization Research Articles

P-419 Relevance of intravenous immunoglobulins in patients with unexplained recurrent pregnancy loss that occurred at the same gestational weeks every time

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent pregnancy loss (RPL) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time, may improve pregnancy outcomes in RPL patients. What is known already There is no established treatment for patients with unexplained RPL. Some investigators have indicated that IVIG may exhibit therapeutic efficacy in women with unexplained RPL. Since it has been reported that live birth rate significantly increased in women with four or more RPL of unexplained etiology, who received a high dose IVIG at 4-5 weeks of gestation in 2022, ESHRE Guideline Group on RPL (2022) has changed the recommendation to the following: the use of repeated and high doses of IVIG very early in pregnancy may improve live birth rate in women with four or more unexplained RPL. Study design, size, duration We performed a retrospective study between September 2013 to March 2023 in two fertility centers. We included 164 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RPL work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) injection was performed twice before and after 1 week of GWL. We divided 164 patients into IVIG group and non-IVIG group by their own choice. The primary outcome was live birth rate, and the secondary outcome was modified live birth rate excluding pregnancies with abnormal fetal karyotype, maternal and neonatal complications. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance IVIG group (n = 116) and non-IVIG group (n = 48) showed no significant differences in the background of patients on BMI (20.3±3.7 and 21.1±2.8), age (37.4±4.2 and 36.7±3.2) and Childbirth history (20.7% and 23.0%), respectively (average±SD), except for the history of past miscarriages. Average numbers of previous miscarriages on IVIG group and non-IVIG group were 3.2±1.3 and 2.5±0.8 (p < 0.001), respectively. After multivariate analysis for BMI, age, Childbirth history and previous miscarriages, live birth rate of IVIG group (70.7%, 82/116) was significantly higher than that of non-IVIG group (60.4%, 29/48; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Similarly, modified live birth rate of IVIG group (73.9%, 82/111) was significantly higher than that of non-IVIG group (63.0%, 29/46; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Treatment outcomes when IVIG was used for each number of previous miscarriages, the modified birth rate was 88.3% for two times miscarriages, 77.0% for three times, and 57.1% for four or more miscarriages. Maternal complications were observed in 2 cases in IVIG group (chest tightness, obstetric critical bleeding). There were no significant differences in the birth weight, gestational weeks, preterm birth, or fetal growth restriction of newborns between two groups. Limitations, reasons for caution Since retrospective nature and we did not perform POC tests for all patients whose pregnancies had ended in miscarriage, we could not assert the effectiveness of IVIG. We need to conduct randomized controlled trial using placebo group to demonstrate the IVIG efficacy before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RPL patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number NA

P-494 Relevance of intravenous immunoglobulins in patients with unexplained recurrent miscarriages that occurred at the same gestational weeks every time

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent miscarriages (RM) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time,may improve pregnancy outcomes in RM patients. What is known already Treatment for patients with unexplained RM are challenging. Since the efficacy of immunotherapy with paternal mononuclear cells has been denied in 1999, some treatment options (e.g., intralipid, G-CSF) have been published with conflicting results. In terms of IVIG, the same story has been described. Because previous studies for IVIG treatment included very heterogeneous group of patients, different dose, different intervals of IVIG, and different starting time of infusions. Recently, a double-blind, randomized, placebo-controlled study for unexplained RM women has been published in 2022, which revealed that IVIG (0.4g/kg) for five consecutive days at 4-5 weeks of gestation improved pregnancy outcomes. Study design, size, duration We performed a retrospective study between September 2013 and May 2021 in two fertility centers. We included 106 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RM work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) was injected twice before and after 1 week of GWL. If the GWL was 8 weeks of gestation, IVIG should be injected 7 and 9 weeks. We defined successful IVIG treatment as live birth. When the pregnancy ended in miscarriage, POC was performed as much as possible. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance Of 106 patients, average age was 37.5 years (28-47 years) and the mean number of previous miscarriages was 3.1. Total 128 cycles of IVIG attempt resulted in 90 (70.3%) successful pregnancies, 36 miscarriages and 2 biochemical pregnancies. After excluding abnormal karyotype of POC (N = 7), 68.6% (83/121) was successful. Per patients, 81.1% (86/106) had at least one live birth. Of 32 patients who experienced miscarriages at the first IVIG attempt, 16 patients got pregnant. Following second IVIG attempt resulted in 12 live births (75.0%). The average weight of live birth babies was 3038g and the birth week was 38.8 weeks of gestation (34-41 wk) for singleton pregnancy (N = 83), whereas 2529g with 35.9 weeks of gestation (33-37 wk) for twin pregnancy (N = 7). Four (4/83, 4.8%) and five (5/7, 71.4%) preterm deliveries were observed for singleton and twin pregnancy, respectively. One baby showed pulmonary hemorrhage resulting in hospitalization in the Neonatal Intensive Care Unit, other live birth babies were without any abnormal findings. Hypertensive disorders of pregnancy were noted in two women resulting in cesarian section delivery. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only two fertility centers. In future clinical research, it is necessary to conduct randomized controlled trials using placebo group to demonstrate the effectiveness of IVIG before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RM patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number not applicable

Overview of the Safety Profile from Efgartigimod Clinical Trials in Participants with Diverse IgG-Mediated Autoimmune Diseases (P1-5.001)

Overview of the Safety Profile from Efgartigimod Clinical Trials in Participants with Diverse IgG-Mediated Autoimmune Diseases (P1-5.001)

Long-Term Safety, Tolerability, And Efficacy of Efgartigimod in Patients with Generalized Myasthenia Gravis: Concluding Analyses from the ADAPT+ Study (S5.006)

Long-Term Safety, Tolerability, And Efficacy of Efgartigimod in Patients with Generalized Myasthenia Gravis: Concluding Analyses from the ADAPT+ Study (S5.006)

Long-term Efficacy and Safety of Symptom-driven Cyclic Rozanolixizumab Treatment in Patients with Generalized Myasthenia Gravis: A Pooled Analysis of a Phase 3 Study and Two Open-label Extension Studies (P1-5.012)

Long-term Efficacy and Safety of Symptom-driven Cyclic Rozanolixizumab Treatment in Patients with Generalized Myasthenia Gravis: A Pooled Analysis of a Phase 3 Study and Two Open-label Extension Studies (P1-5.012)

Efficacy of Rozanolixizumab in Generalized Myasthenia Gravis: Subgroup Analyses from the Randomized Phase 3 MycarinG Study (S5.007)

Efficacy of Rozanolixizumab in Generalized Myasthenia Gravis: Subgroup Analyses from the Randomized Phase 3 MycarinG Study (S5.007)

Dose Selection and Clinical Development of Efgartigimod PH20 Subcutaneous in Patients with Generalized Myasthenia Gravis (P1-5.017)

Dose Selection and Clinical Development of Efgartigimod PH20 Subcutaneous in Patients with Generalized Myasthenia Gravis (P1-5.017)

Achievement of Improved Post-Intervention Status in Patients with Generalized Myasthenia Gravis Treated with Ravulizumab during the CHAMPION MG Study (S5.008)

Achievement of Improved Post-Intervention Status in Patients with Generalized Myasthenia Gravis Treated with Ravulizumab during the CHAMPION MG Study (S5.008)

Intravenous immunoglobulin treatment in women with four or more recurrent pregnancy losses: A double-blind, randomised, placebo-controlled trial.

SummaryBackgroundThere is no effective treatment for women with unexplained recurrent pregnancy loss (RPL). We aimed to investigate whether treatment with a high dose of intravenous immunoglobulin (IVIG) in early pregnancy can improve pregnancy outcomes in women with unexplained RPL.MethodsIn a double-blind, randomised, placebo-controlled trial, women with primary RPL of unexplained aetiology received 400 mg/kg of IVIG daily or placebo for five consecutive days starting at 4–6 weeks of gestation. They had experienced four or more miscarriages except biochemical pregnancy loss and at least one miscarriage of normal chromosome karyotype. The primary outcome was ongoing pregnancy rate at 22 weeks of gestation, and the live birth rate was the secondary outcome. We analysed all women receiving the study drug (intention-to-treat, ITT) and women except those who miscarried due to fetal chromosome abnormality (modified-ITT). This study is registered with ClinicalTrials.gov number, NCT02184741.FindingsFrom June 3, 2014 to Jan 29, 2020, 102 women were randomly assigned to receive IVIG (n = 53) or placebo (n = 49). Three women were excluded; therefore 50 women received IVIG and 49 women received placebo in the ITT population. The ongoing pregnancy rate at 22 weeks of gestation (31/50 [62·0%] vs. 17/49 [34·7%]; odds ratio [OR] 3·07, 95% CI 1·35–6·97; p = 0·009) and the live birth rate (29/50 [58·0%] vs. 17/49 [34·7%]; OR 2·60, 95% CI 1·15–5·86; p = 0·03) in the IVIG group were higher than those in the placebo group in the ITT population. The ongoing pregnancy rate at 22 weeks of gestation (OR 6·27, 95% CI 2·21–17·78; p < 0·001) and the live birth rate (OR 4·85, 95% CI 1·74–13·49; p = 0·003) significantly increased in women who received IVIG at 4–5 weeks of gestation as compared with placebo, but these increases were not evident in women who received IVIG at 6 weeks of gestation. Four newborns in the IVIG group and none in the placebo group had congenital anomalies (p = 0·28).InterpretationA high dose of IVIG in very early pregnancy improved pregnancy outcome in women with four or more RPLs of unexplained aetiology.FundingThe Japan Blood Products Organization.

Hikoboshi Study: Remaining Burden and Current Medical Care Status of Patients with Congenital Hemophilia a: A Study Using Two Japanese Medical Information Databases

Introduction:FVIII prophylaxis for hemophilia A (HA) has reduced the bleeding frequency in patients and enabled the prevention of hemophilic arthropathy and severe bleeding. However, the treatment/disease burden remains a concern, as evidenced by requiring intravenous injections two/three times a week; regular hospital visits to receive a prescription for medication, as drug delivery is not a provision in Japan; and frequent hospital visits because of complications, like bleeding. Moreover, there is no national patient registry for hemophilia in Japan, and few studies have examined the state of medical care and patient burden due to symptoms and medical practices. Herein, data from medical information databases (DBs) were assessed to investigate the state of medical care and patient burden for HA patients in Japan.Methods:The DBs of health insurance subscribers provided by JMDC Inc. (JMDC-DB) and of electronic medical care records provided by Real World Data Co., Ltd. (RWD-DB) were reviewed. Two DBs were used because the differences between them, such as data source for HA patients, may lead to intergroup differences in the characteristics and traceability of each patient.The targets of analysis were HA patients (ICD-10 code: D66) prescribed FVIII concentrates, emicizumab, or bypassing agents. The definition of targets in this analysis was deemed appropriate in a separate validation study.The occurrence rates of intracranial hemorrhage, ischemic heart disease, hospitalizations, emergency visits, and outpatient visits were calculated to evaluate patient burden. Further, data on the number of hospitals visited and prescribed amounts of FVIII concentrates per month were tabulated and descriptive statistics, applied. Table 1 shows the outcome measures.Results:The number of patients included, based on the target period and inclusion criteria, was 459 from JMDC-DB (January 2005 to March 2020) and 229 from RWD-DB (January 1985 to March 2020). Both DBs had a large proportion of patients aged 0-9 years (23.09% in JMDC-DB and 47.16% in RWD-DB) and a small proportion of patients aged ≥60 years (2.61% in JMDC-DB and 5.68% in RWD-DB).The mean (standard deviation [SD]) and median values for the monthly prescribed amount of FVIII concentrate were 10526.36 IU (11260.42) and 8594.91 IU in JMDC-DB and 12569.11 IU (54846.02) and 1514.35 IU in RWD-DB, respectively. The yearly trends of monthly prescribed amounts of FVIII concentrate for patients in JMDC-DB were analyzed. The median values for every 3 years since 2007 were as follows: 1916.67 IU (2007), 3375.00 IU (2010), 7229.17 IU (2013), 8614.58 IU (2016), and 10000.00 IU (2019), indicating an increasing trend in recent years.The occurrence rates (95% confidence intervals [CIs]) of intracranial hemorrhage, ischemic heart disease, and hospitalizations were 2.61% (1.36-4.52), 0.00%, and 32.68% (28.40-37.18) in JMDC-DB and 4.42% (2.14-7.99), 0.44% (0.01-2.44), and 57.08% (50.35-63.62) in RWD-DB, respectively. The age-stratified occurrence rates (95% CIs) of intracranial hemorrhage in JMDC-DB and RWD-DB were 9.43% (4.62-16.67) for 0-9 years, 1.67% (0.04-8.94) for 40-49 years, and 4.00% (0.10-20.35) for 50-59 years and 3.70% (1.02-9.21) for 0-9 years, 13.04% (2.78-33.59) for 10-19 years, 9.09% (1.12-29.16) for 30-39 years, and 6.67% (0.17-31.95) for 40-49 years, respectively. The overall occurrence rates of intracranial hemorrhage were similar in the two DBs: 2.61% (JMDC-DB) vs. 4.42% (RWD-DB).Additional results of our analysis will be presented at the conference.Conclusions:The prescription of FVIII concentrates for Japanese patients with HA is increasing, probably because FVIII prophylaxis in the clinical setting has become more common. The widespread use of FVIII has many benefits for HA patients. However, there are still treatment burdens that should be considered, such as the need for several drug prescriptions and severe bleeding, such as intracranial hemorrhage. [Display omitted] DisclosuresNagao: CHUGAI PHARMACEUTICAL CO., LTD.: Consultancy, Honoraria, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Honoraria, Research Funding; Bayer Yakuhin, Ltd.: Honoraria, Research Funding; Sanofi K.K.: Honoraria; Fujimoto Pharmaceutical Corporation: Honoraria; KM Biologics Co., Ltd.: Honoraria; Pfizer Japan Inc.: Honoraria; Japan Blood Products Organization: Honoraria; Novo Nordisk Pharma Ltd.: Honoraria; CSL Behring K.K.: Honoraria. Ioka: Chugai Pharmaceutical, Co., Ltd.: Current Employment. Nakamura: Chugai Pharmaceutical Co., Ltd.: Current Employment. Murakami: Chugai Pharmaceutical Co., Ltd.: Current Employment. Makishima: Chugai Pharmaceutical, Co., Ltd.: Current Employment. Okada: Chugai Pharmaceutical Co., Ltd.: Current Employment. Sakai: Bayer Yakuhin, Ltd.: Consultancy, Speakers Bureau; Novo Nordisk Pharma Ltd.: Consultancy, Speakers Bureau; CSL Behring K.K.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; CHUGAI PHARMACEUTICAL CO., LTD.: Speakers Bureau; Sanofi K.K.: Speakers Bureau.

Orihime Study: Real World Treatment Patterns and Clinical Outcomes of 338 Patients with Acquired Hemophilia a from a Japanese Health Claims Database

Orihime Study: Real World Treatment Patterns and Clinical Outcomes of 338 Patients with Acquired Hemophilia a from a Japanese Health Claims Database

A Phase II Study of THP-COP (pirarubicin, cyclophosphamide, vincristine and prednisolone) in Patients with Newly Diagnosed Advanced Peripheral T-Cell Lymphoma: THP-3 Study of Japan Hematopoietic Malignancy Clinical Study Group (HMCSG)

BackgroundAlthough CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) has been established as a standard of care in patients (pts) with newly diagnosed peripheral T-cell lymphoma (PTCL), new treatment regimen or strategy has been awaited since the outcome of pts with PTCL treated with CHOP is still poor. Pirarubicin (THP) is, an anthracycline drug, which is not affected by the multidrug resistance mediating p-glycoprotein, and is less cardiotoxic than doxorubicin. THP exhibits activity against some doxorubicin-resistant cell lines.ObjectiveA multicenter phase II study was conducted to evaluate the efficacy and toxicity of THP-COP (pirarubicin, cyclophosphamide, vincristine and prednisolone) therapy in pts with newly diagnosed PTCL by Japan Hematopoietic Malignancy Clinical Study Group.Patients and MethodsEligibility criteria were as follows; newly diagnosed PTCL including PTCL-not otherwise specified (NOS), anaplastic large cell lymphoma (ALCL) and angioimmunoblastic T-cell lymphoma (AITL); clinical stage of bulky II, III or IV; aged 20 to 79 years; PS 0-2 by ECOG's scale. THP-COP consists of pirarubicin (50mg/m2 on Day1), cyclophosphamide (750mg/m2 on Day1), vincristine (1.4mg/m2 max. 2mg, on Day 1) and prednisolone (100mg on Days 1 to 5) every 3 weeks up to 8 cycles in younger pts less than 69 years, and 6 cycles in elderly pts aged 70 to 79 years, The primary endpoint was complete response rate (%CR) including unconfirmed CR (CRu). The planned sample size was 40 pts, which provided at least 80% power with the expected %CR of 55%, threshold of 35%, and a one-sided α of 5%.ResultsFrom October 2007 to December 2012, 41 pts with a median age of 63 (21-77) yrs were enrolled. The numbers of pts with PTCL-NOS, AITL and ALCL was 21 (51%), 14 (34%) and 6 (15%), respectively. The numbers of stage II bulky, III and IV was 2 (5%), 22 (54%) and 17 (41%), respectively.Fifty-four % were International Prognostic Index high/intermediate or high risk. The %CR including %CRu and overall response rate were 51.2% (90% CI; 37.4-64.9) which met the primary endpoint, and 70.7% (95% CI; 54.5-83.9), respectively. The progression-free survival and overall survival at 3 years (3-yr PFS and 3-yr OS) were 43.5% (95% CI; 27.5-58.4) and 62.9% (95% CI; 43.3-77.4), respectively. Most common Grade 4 toxicities were hematologic: %Grade 4 neutropenia and thrombocytopenia were 80.5% and 9.8%, respectively. Cardiac adverse events were observed in 2 patients with Grade 1 arrhythmia. No secondary malignancies were observed.ConclusionsIn pts with untreated advanced PTCL including PTCL-NOS, AITL and ALCL, THP-COP demonstrated high efficacy with durable PFS and OS and acceptable toxicity profiles. Further large-scaled randomized study to compare with CHOP is warranted. DisclosuresOgura:Celgene: Consultancy, Honoraria; Mundipharma: Consultancy; Takeda: Consultancy, Honoraria; Astrazeneca: Honoraria; SymBio: Consultancy, Research Funding; Celltrion, Inc: Consultancy, Honoraria; MeijiSeika Pharma: Consultancy. Ohmachi:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Pfizer Inc: Honoraria; Takeda Pharmaceutical Co., Ltd: Honoraria. Suzuki:MundiPharma: Consultancy; Chugai Pharmaceutical: Honoraria; Sawai Pharmaceutical: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Mochida Pharmaceutical: Honoraria; MSD: Honoraria; Jazz Pharmaceuticals: Consultancy; Kyowa-Hakko Kirin: Honoraria; Shionogi: Honoraria; Meiji Seika Pharma: Honoraria; Ohtsuka: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Takeda Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Gilead Sciences: Consultancy. Atsuta:Otsuka Pharmaceutical Co., Ltd.: Honoraria. Ohyashiki:Chugai: Research Funding; Dainippon Sumitomo: Honoraria; Nippon Shinyaku: Honoraria; Celegen: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Novartis pharma: Honoraria; Novartis: Research Funding; Asteras: Research Funding; Taiho pharmaceutical: Honoraria; Alexion: Honoraria; MSD: Honoraria; Otsuka pharmaceutical: Honoraria; Jansen Pharma: Honoraria, Research Funding; Pfizer: Honoraria; Fujirebio: Honoraria; KyowaHakko Kirin: Honoraria, Research Funding. Hidaka:Chugai: Research Funding; Eisai: Honoraria. Ando:NIHON PHARMACEUTICAL: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Takeda: Other: Donation to institute; ALEXION: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; MSD: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; Eisai: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; CHUGAI PHARMACEUTICAL: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; NOVARTIS: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute. Fukuhara:Nihon Ultmarc, Astellas, AbbVie, Alexionpharma, Bayer Yakuhin, Bristol-Myers Squibb, Baxalta, Celgene, Chugai, Daiichi-Sankyo, Toehringer Ingelheim, Eisai, GlaxoSmithKline, Janssen, Japan Blood Products Organization, Kyowa Hakko Kirin, Mitsubishi Tanabe, : Research Funding; Eisai, Janssen, Takeda, Ono and Zenyaku Kogyo: Honoraria. Morishita:Celgene: Honoraria; Kyowa Hakko Kirin: Honoraria. Tsukasaki:Takeda: Honoraria, Research Funding; Kyowa-Kirin: Honoraria; DaiichiSankyo: Consultancy; Zenyaku Kogyo: Honoraria; Celgene: Honoraria, Research Funding; Mundypharma: Research Funding; HUYA: Honoraria; Chugai/Roche: Honoraria. Ozawa:Kyowa Hakko Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria. Hotta:SymBio: Consultancy; SellSeed: Other: outside director; Jansen Pharma: Honoraria; Meiji Seika Pharma: Honoraria; Zenyaku Kogyo: Honoraria. Kinoshita:Chugai: Honoraria, Research Funding; Ono: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Zenyaku: Honoraria, Research Funding; Jansen Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Eisai: Honoraria; Bristol-Meier's-Squib: Honoraria; Solaisia: Research Funding; MSD: Research Funding; Takeda: Research Funding.

Highly Sensitive Viral RNA-Seq Is a Powerful Tool to Analyze the Viral Transcripts in HTLV-1-Infected Individuals

IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus which causes two major pathologies in infected individuals, Adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There are tens of thousands of different infected clones in asymptomatic HTLV-1 carriers (AC) and each clone can be identified by the viral integration site. Monoclonal integration of HTLV-1 provirus is observed in the host genomic DNA in ATL cells, suggesting that these originate from a single virus-infected clone. It is well known that Tax and HBZ, encoded in the sense and antisense strands of HTLV-1 provirus, respectively, have the ability to induce oncogenesis. The question is how a certain infected clone, among various different ones, becomes a malignant clone. One hypothesis is that a malignant clone may express viral gene transcripts, associated with oncogenesis, more abundantly than a non-malignant clone. Another hypothesis is that HTLV-1 integration may induce an abnormal transcript in the host cell, such as the aberrant expression of the host gene nearby the integration site or the generation of a chimeric transcript between the virus and the host. To analyze HTLV-1 transcript or explore the presence of virus-host chimeric transcripts efficiently, we aimed to establish a method with higher resolution than the conventional RNA-seq by using DNA probe targeting for the entire HTLV-1 sequence. And then, we applied the protocol to characterize the pattern of transcription from HTLV-1 provirus and analyzed virus-host chimeric transcripts in patients' samples.MethodsTo check the efficiency and possible bias of the highly sensitive viral RNA-seq analysis, we analyzed viral transcript of an HTLV-1-infected clone, TBX-4B. Subsequently, mRNA-seq was performed with PBMCs from 6 AC, 4 HAM/TSP, and 5 ATL patients. After cDNA libraries synthesis, HTLV-1 sequences were enriched by the probe-based capture method as we reported previously for DNA-seq (Miyazato et al, Sci Rep 2016). The enriched libraries were sequenced in an Illumina NextSeq device.ResultsWe analyzed HTLV-1 transcript of a virus-infected T-cell clone both before and after the enrichment by viral-specific DNA probes. The proportion of viral sequences within the total number of reads was significantly increased from 0.05% to 80.62% (1600-fold) by the enrichment method. We found that most of the viral regions were significantly and similarly enriched by the additional enrichment step, suggesting the DNA probe-based enrichment of HTLV-1 sequences is applicable to RNA-seq. Next, we applied the protocol to analyze PBMC samples from HTLV-1-infected individuals. The results showed that the level of anti-sense HTLV-1 transcription was significantly higher than that of the sense transcription, not only in ATL patients, which is consistent with previous reports by other groups, but also in AC and HAM/TSP patients. Average proportion of sense and antisense signals were 3.42% and 96.46%, respectively. Even after the enrichment step, tax expression, a sense viral transcript, was undetectable in most of the samples except for one AC and one ATL patient, while an anti-sense transcript HBZ was detectable in all infected individuals. There was no significant difference in the expression level of HBZ between different clinical states. We identified more abundant virus-host chimeric transcripts in ATL samples than in HAM/TSP patients or AC. In ATL cases, HTLV-1 generated chimeric transcripts with the following host genes, HSD17B11, COLEC12, AHSA2, EEF2, TAOK1, KIAA0100, and KIF1B . The presence of abundant chimeric transcripts was validated by RT-PCR.DiscussionWe have established a highly sensitive viral RNA-seq for HTLV-1 transcript analysis. That significantly increased the sensitivity of detection of proviral and chimeric transcripts between virus and the host genome. The result was not consistent with the hypothesis that a malignant clone may express viral gene transcripts more abundantly than a non-malignant clone. Thus, the presence of chimeric transcripts could play a role in cell proliferation and help us reveal the mechanism of clonal selection for HTLV-1-infected cells. We are now going to analyze virus-host chimeric transcripts detected by the high sensitive RNA-seq in more patients' samples to clarify their association with oncogenesis. [Display omitted] DisclosuresYamano:Daiichi Sankyo: Research Funding; Ono Pharma: Research Funding. Utsunomiya:Celgene: Honoraria; Novartis Pharma: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria; Chugai Pharma: Honoraria; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Nippon Shinyaku: Honoraria; HUYA Bioscience International: Honoraria; Pfizer: Honoraria.

Successful MPB Therapy Followed By Bortezomib Maintenance for Non-Transplant Eligible Patients with Newly Diagnosed Multiple Myeloma: Japanese Phase 2 Study - Broad-J Study

[BACKGROUND]Although recent novel agents remarkably improve overall survival (OS) for multiple myeloma (MM), intensive chemotherapy can induce severe adverse events (AEs), resulting in a high discontinuation rate, especially in non-transplant eligible (NTE) patients with MM. Although modification of therapy can be focused on, standard management needs to be investigated. To explore this issue, we designed a Japanese phase 2 clinical single arm study, BoRtezomib-based Optimized therapy Aiming Disease control in Japan (BROAD-J study), composed of a weekly MPB as induction therapy followed by bortezomib maintenance therapy (Bor-MT) every 2 weeks or less for newly-diagnosed (ND) symptomatic NTE-MM patients.[PATIENTS AND METHOD]Patient eligibility/exclusion criteria: Patients aged ≥ 20 years with ND-MM, having measurable disease and a Karnofsky Performance Status (KPS) ≥ 60%, were enrolled. The patients were symptomatic and ineligible for autologous stem cell transplantation. The diagnosis of MM was made by IMWG criteria. Exclusion criteria were a history of allergy against Bor, mannitol, or boron; having an active cancer(s); positivity for hepatitis B antigen or hepatitis C antibody; severe abalienation; severe pulmonary interstitial change; severe active infection, severe pulmonary dysfunction or severe heart dysfunction; the presence or possibility of pregnancy.Study design: This phase 2 study enrolled 84 patients from December 2011 to February 2013. The induction phase consisted of modified MPB: melpharan on days 1 and 4 (6 mg/m2 p.o), every 35-day cycle, and prednisolone on days 1-4 (40 mg/day, p.o., every 35-day cycle; Bor: 1.3 mg/m2, i.v., d1, 8, 15, 22 every 35-day cycle). The number of induction cycles was not fixed. After achieving a partial response (PR) or better, Bor-MT was started based on the physicians' decision. Bor-MT consisted of twice or less often per month of Bor (1.3 mg/m2, i.v.) with/without additional drugs such as dexamethasone, until progressive disease developed. A dose reduction of Bor was permitted to 1.0 or 0.7 mg/m2 based on AEs or the physicians' decision. The primary objective was the continued treatment duration and adverse event rate. Secondary objectives included maximum response, overall survival, and time to progression duration. AEs were graded according to NCI-CTCAE v4.0. All patients provided written informed consent, which was conducted in accordance with the Declaration of Helsinki.[RESULTS]Median age was 74 years (60-86), and the sex ratio was 36:46 (M:F). The subtypes of MM were IgG 54, IgA 12, IgD 1 and BJP 15, and ISS was I 13, II 35, and III 34. The ratio of performance status of 3 and 4 was 20%. The median observed duration was 4.3 years (0.1-5.2). Sixteen patients (20%) are still on study, and the median treatment duration, one of the primary endpoints, was 1.6 years (0.9-5.1). The median MPB induction cycles were 4 (1-9). Sixty-five patients (79%) were moved to Bor-MT, and the median Bor-MT duration was 1.6 years (0.1-4.9). Regarding the best response, ratios of ≥ VPGR and ≥ PR were 38.8% and 82.5%, respectively. In the induction phase, 46.5% of patients had ≥ grade 3 AEs, whereas 16.1% in the Bor-MT. Fifty-nine patients were alive and 23 patients were dead at the time of writing. The median OS was not reached, and 5-year OS was 61.8%.[CONCLUSION]Although the majority of enrolled patients were older (the median age was 74 years), this study found a similar or better outcome compared to those of previous MPB studies. Reduced doses of MP and careful Bor-MT administration were performed in this study, resulting in a better OS and response rate among the ND-NTE-MM patients. [Display omitted] DisclosuresTokuhira:Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Imai:Jansen Pharma,: Honoraria. Ishida:Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Takeda: Honoraria; ONO: Honoraria; Celgene: Honoraria. Nakazato:Nippon Shinyaku: Honoraria; Sumitomo Dainippon Pharma: Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Fujimoto Pharmaceutical: Honoraria; Celgene: Honoraria; Janssen Pharmaceutical: Honoraria; Takeda Pharmaceuticals: Honoraria. Mitani:Kyowa Hakko Kririn Co., Ltd: Research Funding; Novartis Pharma K.K.: Research Funding; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kririn Co., Ltd: Speakers Bureau; Chugai Pharmaceutical Co., Ltd: Research Funding; TEIJIN PHARMA LIMITED: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Ohyashiki:Novartis pharma: Honoraria; Fujirebio: Honoraria; Pfizer: Honoraria; Jansen Pharma: Honoraria, Research Funding; Otsuka pharmaceutical: Honoraria; Taiho pharmaceutical: Honoraria; Alexion: Honoraria; MSD: Honoraria; KyowaHakko Kirin: Honoraria, Research Funding; Chugai: Research Funding; Novartis: Research Funding; Asteras: Research Funding; Bristol-Myers Squibb: Honoraria; Celegen: Consultancy, Honoraria; Nippon Shinyaku: Honoraria; Dainippon Sumitomo: Honoraria. Suzuki:Ono: Honoraria; Fujimoto: Honoraria; Novarltis: Honoraria; Takeda: Honoraria; Celgen: Honoraria; Bristol-Myers K.K: Honoraria.

First-in-Human Study of the EZH1/2 Dual Inhibitor DS-3201b in Patients with Relapsed or Refractory Non-Hodgkin Lymphomas — Preliminary Results

First-in-Human Study of the EZH1/2 Dual Inhibitor DS-3201b in Patients with Relapsed or Refractory Non-Hodgkin Lymphomas — Preliminary Results

Randomized Phase II Study of R-CHOP-14 Versus R-CHOP-14 Followed By Chaser As Induction Therapy for High-Dose Chemotherapy (HDT), LEED, and Autologous Stem-Cell Transplantation (ASCT) in Poor-Risk Diffuse Large B-Cell Lymphoma (DLBCL): Japan Clinical Oncology Group (JCOG) Study (JCOG0908)

Randomized Phase II Study of R-CHOP-14 Versus R-CHOP-14 Followed By Chaser As Induction Therapy for High-Dose Chemotherapy (HDT), LEED, and Autologous Stem-Cell Transplantation (ASCT) in Poor-Risk Diffuse Large B-Cell Lymphoma (DLBCL): Japan Clinical Oncology Group (JCOG) Study (JCOG0908)

Improved Prognosis of Extranodal NK/T-Cell Lymphoma, Nasal Type (ENKL) of Nasal Origin but Not Extranasal Origin: An Analysis of NKEA Study

Improved Prognosis of Extranodal NK/T-Cell Lymphoma, Nasal Type (ENKL) of Nasal Origin but Not Extranasal Origin: An Analysis of NKEA Study

Phase 1/2 Study Evaluating the Safety, Pharmacokinetics, and Preliminary Efficacy of Venetoclax in Japanese Subjects with Chronic Lymphocytic Leukemia

Background: Venetoclax (VEN) is an orally bioavailable, small molecule inhibitor of BCL-2, an important anti-apoptosis regulator that is commonly overexpressed in chronic lymphocytic leukemia (CLL). In previous studies, VEN demonstrated acceptable safety and antitumoral activity in the treatment of hematologic malignancies.Methods: This arm of an open-label, multicenter, phase 1/2 study (NCT02265731) was designed to evaluate the safety, pharmacokinetics (PK), and preliminary efficacy of VEN in Japanese patients with relapsed or refractory (R/R) CLL or small lymphocytic leukemia (SLL). Main inclusion criteria were age ≥20 years, Eastern Cooperative Oncology Group performance score ≤1, and CLL or SLL that was R/R to standard treatment (eg, fludarabine- or alkylator-based regimen). Patients could not have undergone allogeneic or autologous stem cell transplantation. VEN monotherapy was started at a dosage of 20 mg/day given orally on Week 1 Day 1. If a patient developed electrolyte changes suggestive of tumor lysis syndrome (TLS), VEN was withheld until the changes resolved. Otherwise, the dosage of VEN was increased, as tolerated, to 50 mg/day in Week 2, to 100 mg/day in Week 3, to 200 mg/day in Week 4, and to 400 mg/day in Week 5 and afterward. Adverse events (AEs) and laboratory values were monitored for safety. Dose-limiting toxicities (DLTs) were determined during the step-up period (if applicable) until the completion of Week 7. Efficacy was measured as the overall response rate, complete remission (CR), and partial remission (PR).Results: Six patients were enrolled: 4 with CLL and 2 with SLL. Their median age was 72 years (range, 38-73 years), and 4 (67%) were male. As of July 7, 2017, all 6 patients achieved objective responses, which included 1 CR (in a patient with 17p deletion CLL) and 5 PRs. No DLTs were reported, and no cases of laboratory or clinical TLS occurred during dosing ramp-up. Based on the preliminary analysis, average VEN exposure at the 400-mg dose was 20-30% higher in Japanese patients with CLL than in Western patients with CLL, but individual PK exposures were within the range of values previously reported in non-Japanese patients with CLL (Table). AEs of any grade possibly related to VEN (occurring in ≥2 patients) were neutropenia (n=6), lymphopenia (n=5), thrombocytopenia (n=2), and vomiting (n=2). AEs of grade ≥3 possibly related to study drug were neutropenia (n=5), lymphopenia (n=3 patients), myelodysplastic syndrome (n=1, serious AE), and disseminated Herpes zoster infection (n=1, serious AE). Other serious AEs, thought to be unrelated to study drug, were febrile neutropenia, pneumonia, septic shock, and large intestinal ulcer (all n=1). The large intestinal ulcer led to the only interruption of VEN. No deaths have occurred.Conclusions: In Japanese patients with R/R CLL or SLL, VEN as monotherapy demonstrated a tolerable safety profile and produced an objective response in all 6 patients. The VEN PK exposure range was similar to that previously observed in non-Japanese patients. [Display omitted] DisclosuresHatake:AbbVie, Gilead, Celgene, Solasia, Pfizer, Bristol-Myers Squibb, Janssen, Ghugai: Research Funding; Mundipharma K.K.: Honoraria. Yamamoto:AbbVie, Celgene, Ono Pharmaceutical, ARIAD Pharmaceuticals, Novartis, Takeda, Eisai, Solasia, MSD, Chugai, Gilead Sciences: Research Funding; ARIAD Pharmaceuticals/CMIC, Celgene, Novartis, Pfizer, Otsuka Pharmaceutical, Takeda, Kyowa Hakko Kirin, Bristol-Myers Squibb, Sumitomo Dainippon Pharma, Ono Pharmaceutical, Mundipharma, Chugai: Honoraria; Ono Pharmaceutical, Meiji Seika Pharma, Novartis, Otsuka Pharmaceutical, Mundipharma, Boehringer Ingelheim: Consultancy. Fukuhara:Nihon Ultmarc, Astellas, AbbVie, Alexionpharma, Bayer Yakuhin, Bristol-Myers Squibb, Baxalta, Celgene, Chugai, Daiichi-Sankyo, Toehringer Ingelheim, Eisai, GlaxoSmithKline, Janssen, Japan Blood Products Organization, Kyowa Hakko Kirin, Mitsubishi Tanabe, : Research Funding; Eisai, Janssen, Takeda, Ono and Zenyaku Kogyo: Honoraria. Kusumoto:Chugai: Honoraria, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing support, under the direction of Shigeru Kusumoto, was provided by Cheryl Wright of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd, Research Funding. Izutsu:Abbvie, Astellas, Astellas Amgen, Bayer, Celgene, Celltrion, Chugai, Daiichi Sankyo, Eisai, HUYA Bioscience International, Janssen Edit, Mundhi, Novartis, Ono, Sanofi, Solasia, Symbio, Takeda, Zenyaku: Research Funding; Chugai, Eisai, Gilead, Janssen Edit, Kyowa Hakko Kirin, MSD, Ono, Takeda: Honoraria; Celgene, Bayer: Consultancy. Nagai:Janssen, Mundipharma, Celgene, Bayer Yakuhin, AbbVie, Takeda, Chugai, Kyowa Hakko Kirin, Eisai: Research Funding; Chugai, Mundipharma, Eisai, Sanofi, Janssen: Honoraria. Honda:AbbVie: Employment, Equity Ownership. Saeki:AbbVie: Employment, Equity Ownership. Roberts:AbbVie: Employment, Equity Ownership. Maciag:AbbVie: Employment, Equity Ownership. Agarwal:AbbVie: Employment, Equity Ownership. Salem:AbbVie: Employment, Equity Ownership. Freise:AbbVie: Employment, Equity Ownership. Kiriyama:AbbVie: Employment, Equity Ownership. Tobinai:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; AbbVie: Research Funding; HUYA Bioscience: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Servier: Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria.

EZH1/2, SWI/SNF, and MLL2 Dependent Heterochromatin Formation and Abnormal Transcriptome in Hematological Malignancies

The neomorphic alteration of H3K27me3 patterning is a fundamental trait of cancers. We have developed a new clinical strategy; dual inhibition of EZH1 and EZH2 significantly reprogrammed the abnormal epigenome and restored the transcriptome in lymphomas (Blood, 2016; 58th ASH, 2016; NCT02732275).H3K27me3-associated chromatin regulation contributes to almost all human malignancies, yet the underlying mechanisms of gene silencing remain unclear, owing to a lack of data representing the action of EZH1/2 and other chromatin regulators in practical models. To analyze the dynamic processes of chromatin regulation, we treated H3K27me3-accumulated lymphoma cells with EZH2 inhibitors or EZH1/2 dual inhibitors and then profiled several epigenetic factors and histone marks using time-course chromatin immunoprecipitation (ChIP) and genome-wide ChIP assays. EZH2 single inhibition expelled the EZH2-containing polycomb repressive complex 2 (EZH2-PRC2) from methylated chromatin. This simultaneously triggered the acute recruitment of EZH1-PRC2 to a part of the loci, leading to homeostatic regulation of chromatin. In contrast, pharmacological EZH1/2 dual inhibition or EZH1 depletion prior to EZH2 inhibition resulted in the rapid removal of both PRC2 and PRC1 and their enzymatic products H3K27me3 and H2AK119ub. These initial changes were further accompanied by the recruitment of chromatin remolding factors (SWI/SNF, also called BAF complex), such as ARID1A, BRG1 (SMARCA4), and SNF5 (SMARCB1), histone H3K27 acetylation and H3K4 tri-methylation, formation of accessible focal chromatin, RNA PolII recruitment, and stable induction of transcription. We also found that hyperactivation of EZH1/2 by co-overexpression or EZH2 active mutation, as in a part of B-cell lymphoma, facilitated the eviction of the BAF complex and closed chromatin conformation. Thus, the opposing actions of EZH1/2-containing polycomb and SWI/SNF complexes are required for facultative heterochromatin formation.Genetic lesions and transcriptional downregulation of epigenetic factors are frequently observed in general cancers, including hematological malignancies. We reconstructed the loss-of-function (LoF) models of major genetic targets ARID1A (9% in FL, 7% in BL) , SMARCA4 (21% in BL) , KDM6A (found in AML, CML, T-ALL, MM, HL) , and BAP1 and then analyzed them using epigenome/transcriptome profiling. Inactivation of the tumor-suppressive chromatin remodelers caused EZH1- and EZH2-dependent H3K27me3 accumulation and heterochromatin formation, leading to transcriptional silencing. The new EZH1/2 dual inhibitor effectively cancelled epigenetic abnormalities. The cell models lacking a functional BAF complex were sensitive to the EZH1/2 dual inhibitors compared with the EZH2 single inhibitor.MLL2 (KMT2D)-mediated H3K4me3 regulation is frequently disrupted in DLBCL (~30%) and FL (~90%). MLL2 LoF enhanced cell growth in pre-tumorigenic LCLs, which could be cancelled by EZH1/2 dual inhibition. Genome-wide ChIP revealed that MLL2 LoF-dependent H3K4me3 downregulation is closely associated with EZH1- and EZH2-dependent H3K27me3 accumulation. Collectively, the EZH1/2 dual inhibitors may be promising for use in (1) EZH1/2 dual expressing tumors, such as ATL and other T- and B-cell lymphomas; (2) DLBCL/FL with actively mutated EZH2; and (3) additional cancer types (~20% of human malignancies) with mutations in the Trithorax and other epigenetic genes (ARID1A, SMARCA4, SMARCB1, KDM6A, BAP1, and MLL2).The EZH1/2-dependent epigenetic pathway of cancer development is also initiated by viral infection (Curr. Opin. Oncol., 2017). Whole epigenome and transcriptome analyses revealed that infection by lymphotropic oncoviruses, HTLV-1 and EBV, reprogrammed global EZH1/2-H3K27me3 patterns and established unique transcriptomes in malignant progenitors. Low-dose EZH1/2 inhibitor selectively depleted the HTLV-1-infected cell population in asymptomatic carriers (31/35, 88.6%) and EBV-infected immortalized LCLs. Data integration with established lymphoma epigenome suggested that this heritable EZH1/2 dysfunctional state is epigenetically imprinted at the non-malignant, virus-infected step. The identification of this novel epigenetic pathway will lead to precision oncology and new therapeutic strategies in human malignancies, particularly for elimination of the founder cell populations. DisclosuresYamagishi:Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Utsunomiya:HUYA Bioscience International: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Celgene: Honoraria; Astellas Pharma: Honoraria; Pfizer: Honoraria; Nippon Shinyaku: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Novartis Pharma: Honoraria; Chugai Pharma: Honoraria. Tsukasaki:HUYA: Honoraria; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundypharma: Research Funding; Zenyaku Kogyo: Honoraria; Chugai/Roche: Honoraria; Kyowa-Kirin: Honoraria; DaiichiSankyo: Consultancy. Tobinai:AbbVie: Research Funding; HUYA Bioscience: Honoraria; Mundipharma: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Servier: Research Funding; Takeda: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria. Araki:Daiichi Sankyo: Employment. Uchimaru:Daiichi Sankyo Co., Ltd.: Research Funding.

Establishment and Characterization of in Vitro Model of X-Linked Sideroblastic Anemia

(Background) Congenital sideroblastic anemia (CSA) is an inherited microcytic anemia characterized by the presence of bone marrow ring sideroblasts. The most common form of CSA is X-linked sideroblastic anemia (XLSA), which is attributed to mutations in the X-linked gene erythroid-specific 5-aminolevulinate synthase (ALAS2) . ALAS2 encodes the enzyme that catalyzes the first and rate-limiting steps in the heme biosynthesis pathway in erythroid cells. This pathway converts glycine and acetyl-CoA to 5-aminolevulinic acid (ALA), which requires pyridoxal 5'-phosphate (PLP) as a cofactor. Although PLP has been used for treating XLSA, a marked proportion of patients with XLSA remain refractory to treatment. Therefore, to elucidate the details of the underlying molecular mechanisms that contribute to ring sideroblast formation for XLSA, we succeeded to induce ALAS2 gene mutation in human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells by CRISPR/Cas9 (European Hematology Association 2017). Recently we have further extended the study; we succeeded to establish ring sideroblast by newly optimized in vitro co-culture system and conducted its molecular characterization.(Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells. The mutation diminished the binding of transcription factor GATA-1, which lead to decreased transcription of the ALAS2 gene, thereby causing XLSA (Kaneko et al . Haematologica 2014). Co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, doxycycline, MTG, Insulin-Transferrin-Selenium, and L-ascorbic acid. Prussian blue staining and electron microscopy were used to assess iron deposition in erythroblasts. For transcription profiling, Human Oligo chip 25K (Toray) was used. Apoptotic cell was determined with annexin V.(Results) The established XLSA clone harbored 19-bp deletion within the intron 1 enhancer region of ALAS2, including GATA binding domain. The XLSA clone appeared pink/pale color, accompanied by the decreased intracellular heme concentration. Quantitative ChIP analysis demonstrated that the chromatin occupancies of GATA-1 and its cofactor TAL1 were significantly abrogated at the ALAS2 intron 1 loci in the XLSA clone. Quantitative RT-PCR analysis demonstrated significant downregulation of ALAS2 as well as globin genes in the XLSA clone. Microarray analysis revealed >2-fold up- and down-regulation of 619 and 274 genes caused by the 19-bp deletion, respectively. The downregulated gene ensemble included globins as well as genes involved in iron/heme metabolism (ALAS2, TFRC, CPOX, and MFRN1). GO analysis revealed significant enrichment of genes involved in cellular iron ion homeostasis, regulation of transcription, and regulation of apoptosis. In support of the observation, the apoptotic cell frequency was increased in the XLSA clone than the wild-type cells.When the XLSA clone was induced to undergo erythroid differentiation by co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC), a majority of the erythroblasts exhibited aberrant mitochondrial iron deposition (Figure). Intriguingly, the co-culture without SFC nor even with transferrin-loaded iron did not induce ring sideroblasts. By the co-culture with SFC, ALAS2 as well as globin mRNA levels in the XLSA clone was obviously induced to the level comparable to the wild-type cells. Quantitative ChIP-based mechanistic analysis at the ALAS2 locus demonstrated that GATA-1 occupancy was still abrogated at intron 1, while it was significantly increased at intron 8, in the XLSA clone-derived ring sideroblasts. Noticeably, when SFC was supplemented to induce ring sideroblasts, the apoptotic cell frequency was significantly reduced as compared with the XLSA clone co-cultured without SFC. Also, microarray analysis identified upregulation of anti-apoptotic genes (i.e. HSP70) in the ring sideroblast, implying that ring sideroblast formation might be a consequence of cytoprotective reaction. Finally, ALA treatment significantly improved the consequences compromised heme synthesis in the XLSA clone, implying that ALA may represent a therapeutic option for XLSA.(Conclusion) Further characterization of the XLSA model would help to clarify its molecular etiology as well as to establish novel therapeutic strategies. [Display omitted] DisclosuresFukuhara:Eisai, Janssen, Takeda, Ono and Zenyaku Kogyo: Honoraria; Nihon Ultmarc, Astellas, AbbVie, Alexionpharma, Bayer Yakuhin, Bristol-Myers Squibb, Baxalta, Celgene, Chugai, Daiichi-Sankyo, Toehringer Ingelheim, Eisai, GlaxoSmithKline, Janssen, Japan Blood Products Organization, Kyowa Hakko Kirin, Mitsubishi Tanabe, : Research Funding.