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The European Treatment and Outcome Study (EUTOS) long‐term survival (ELTS) score predicts disease‐specific death in patients with chronic myeloid leukemia (CML) being treated with imatinib during the chronic phase (CP) of the disease. However, it is unclear whether the ELTS score predicts CML‐related events or treatment responses. This study evaluated the predictive value of the ELTS score regarding prognosis and treatment response in patients with CML‐CP. Clinical data were retrospectively obtained from patients enrolled in the CML Cooperative Study Group (CML‐CSG), which included patients diagnosed with CML‐CP from April 2001 to January 2016, and treated with any tyrosine kinase inhibitor (TKI) as first‐line therapy. Among 342 eligible patients, the ELTS scores indicated low‐, intermediate‐, and high‐risk in 74%, 21%, and 5% of patients, respectively. Patients with high ELTS scores had significantly higher disease‐specific mortality and worse event‐free survival, progression‐free survival, and overall survival. Among four risk scores, including the Sokal, Hasford, EUTOS, and ELTS scores, risk stratification by the ELTS score had the highest predictive value in assessing patient prognosis, and also in treatment responses. In fact, the EUTOS and ELTS scores were able to predict the major molecular response within 12 months. Most importantly, the ELTS score was the only scoring system that predicted deep molecular response at any time, regardless of risk level (65.0%, 43.7%, and 23.5% in low‐, intermediate‐, and high‐risk groups, respectively). Compared to other risk scores, the ELTS score was the most sensitive risk classification tool for the four endpoints of interest in this study, as well as molecular responses in patients with CML‐CP., In this real‐world TKIs treated CML‐CP patients cohort, the EUTOS long‐term survival (ELTS) score was the most sensitive risk classification tool to predict CML‐specific deaths, EFS, PFS, and OS compared to the Sokal, Hasford, and EUTOS scoring systems. Notably, the ELTS score was the only scoring system that predicted cumulative incidence of deep molecular response.

Regressive lymphoproliferative disorders (R-LPD) after methotrexate (MTX) withdrawal are one of the specific features of methotrexate - associated lymphoproliferative disorders (MTX-LPD). Although the impact of the absolute lymphocyte count (ALC) on the pathogenesis of R-LPD has been recently emphasized, understanding relapse/regrowth events (RRE) and differences among LPD subtypes is necessary. In this study, we confirmed ALC recovery in the regressive group (R-G; R-LPD without RRE) and relapse/regrowth group (R/R-G; R-LPD with RRE). The increase in ALC lasted at least 2 years in R-G, whereas it decreased within 3 years in R/R-G, supporting the better overall survival (OS) in R-G, as previously reported. In addition, our study suggested that an ALC of 1000/µL at the time of development of LPD is a significant predictor for treatment-free survival (TFS). Furthermore, an ALC of 1000/µL at 6 months after MTX withdrawal was found to be a significant indicator of TFS and OS for R-G and R/R-G. The ALC decreased gradually before LPD development in R/R-G, whereas it decreased 6 months before LPD development in R-G, confirming the important role of ALC in the pathogenesis of MTX-LPD such as regressive events and RRE. In addition to ALC, other predictive factors, such as serum C-reactive protein and soluble interleukin-2 receptors, may be helpful in the management of MTX-LPD, including the decision making for an additional chemotherapy for regressive LPD after MTX withdrawal.

Methotrexate (MTX) is one of the potent drugs for autoimmune diseases (ADs), especially for rheumatoid arthritis. Recent studies suggest that MTX should be immediately withdrawn when patients with AD develop lymphoproliferative disorder (LPD). However, biopsy cannot be performed for diagnosis because LPD regresses quickly after MTX withdrawal, thus making clinical MTX-LPD (c-MTX-LPD) challenging to diagnose. In this study, among the 28 patients with c-MTX-LPD, seven developed a proven LPD (p-LPD) after suspicious LPD (s-LPD) regression, six of which were Hodgkin lymphoma. Four of seven patients with p-LPD + died, whereas all patients with p-LPD- survived. The clinical manifestations indicative of p-LPD include fever, elevated serum C-reactive protein level, and soluble interleukin-2 receptor level. Anti-AD drugs did not appear to affect the pathogenesis of p-LPD development. P-LPD was not observed after 3 years from the time of s-LPD regression.

Other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD), a category of immunodeficiency-associated LPD according to the World Health Organization classification, is associated with immunosuppressive drugs (ISDs). Several factors, including autoimmune disease (AID) activity, Epstein-Barr virus (EBV) infection, ISD usage, and aging, influence the development of OIIA-LPD, resulting in complicated clinical courses and outcomes. Most OIIA-LPD develops in patients with rheumatoid arthritis using methotrexate (MTX-LPD). The management of MTX-LPD is based on the clinical course, i.e., with/without regression, with/without relapse/regrowth event (RRE), LPD subtype, and ISDs for AIDs after LPD development. There are three clinical courses after ISD withdrawal: regressive LPD without relapse/regrowth (R-G), regressive LPD with RRE (R/R-G), and persistent LPD (P-G). The majority of EBV+ diffuse large B-cell lymphomas are classified in R-G, whereas classic Hodgkin lymphoma is generally classified in R/R-G. Polymorphic LPD (P-LPD) in MTX-LPD develops with heterogeneous pathological features similar to monomorphic LPD. Chemotherapy for MTX-LPD is selected according to that for de novo LPD, although the strategy for aggressive P-LPD and non-specific LPD is not well established. The absolute lymphocyte count in the peripheral blood has been suggested as a candidate marker for MTX-LPD development and RRE. Several clinical issues, including correct diagnosis among overlapping clinicopathological features in MTX-LPD and clinical management of LPD by ISDs other than MTX, require further investigation.

The purpose of this trial was to evaluate the efficacy of 2-year consolidation therapy with nilotinib, at a dose of 300 mg twice daily, for achieving treatment-free remission in chronic myeloid leukemia patients with a deep molecular response (BCR-ABL1IS ≤0.0032%). Successful treatment-free remission was defined as no confirmed loss of deep molecular response. We recruited 96 Japanese patients, of whom 78 sustained a deep molecular response during the consolidation phase and were therefore eligible to discontinue nilotinib in the treatment-free remission phase; of these, 53 patients (67.9%; 95% confidence interval: 56.4–78.1%) remained free from molecular recurrence in the first 12 months. The estimated 3-year treatment-free survival was 62.8%. Nilotinib was readministered to all patients (n=29) who experienced a molecular recurrence during the treatment-free remission phase. After restarting treatment, rapid deep molecular response returned in 25 patients (86.2%), with 50% of patients achieving a deep molecular response within 3.5 months. Tyrosine kinase inhibitor withdrawal syndrome was reported in 11/78 patients during the early treatment-free remission phase. The treatment-free survival curve was significantly better in patients with undetectable molecular residual disease than in patients without (3-year treatment-free survival, 75.6 versus 48.6%, respectively; P=0.0126 by the log-rank test). There were no significant differences in treatment-free survival between subgroups based on tyrosine kinase inhibitor treatment before the nilotinib consolidation phase, tyrosine kinase inhibitor-withdrawal syndrome, or absolute number of natural killer cells. The results of this study indicate that it is safe and feasible to stop tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia who have achieved a sustained deep molecular response with 2 years of treatment with nilotinib. This study was registered with UMIN-CTR (UMIN000005904).

Our study aims to highlight the critical role of the introduction of second generation tyrosine kinase inhibitors (2nd TKIs) on the prognosis of patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP), as determined by European Treatment and Outcome Study (EUTOS) system. Patients who were diagnosed with CML-CP before March 2009 were classified into the imatinib group, and those diagnosed after April 2009 were classified into the 2nd TKI group. EUTOS high-risk patients exhibited significantly worse outcomes in terms of event-free survival (EFS), progression-free survival (PFS), and CML-associated death than those considered to be low-risk. Risk stratification by EUTOS score was predictive of risk-associated clinical outcomes in patients classified into the imatinib group; however, the EUTOS score failed to predict the outcomes of patients classified into the 2nd TKI group. Our data suggest that the introduction of 2nd TKIs might have improved treatment outcomes, particularly in EUTOS high-risk patients.

This study was performed to investigate the features and outcome of patients with therapy-related chronic myeloid leukemia (TR-CML) who were treated with tyrosine kinase inhibitors (TKIs). The analysis included 308 patients with CML in the chronic phase who were extracted from the CML Cooperative Study Group database. Of these patients, 11 (3.6%) were identified as having TR-CML. No differences in age, sex, white blood cell count, hemoglobin level, platelet count, or European Treatment and Outcome Study risk were observed between patients with TR-CML vs. de novo CML. However, the responses of TR-CML patients to TKIs (6, 3, and 2 received imatinib, nilotinib, and dasatinib, respectively) were excellent; all achieved major or deep molecular response. Furthermore, the outcomes of TR-CML patients were relatively favorable; the 3-year event-free survival rates in the TR-CML and de novo CML patients were 100% and 94%, respectively; the difference was not statistically significant. In conclusion, our study showed that TR-CML patients could achieve a good clinical course with TKI therapy. Detailed investigations of TR-CML may provide new insights into CML biology.

Methotrexate (MTX) carries a risk of lymphoproliferative disorders (LPDs), but MTX-associated LPDs (MTX-LPDs) can resolve spontaneously after MTX withdrawal. However, the precise clinicopathologic features of MTX-LPD remain unclear. We aimed to investigate the clinicopathologic characteristics, outcomes, and prognostic factors for histologic types of MTX-LPD. Paraffin-embedded tissue samples of 219 patients with MTX-LPD were analyzed. In total, 30,33,106, and 26 had reactive lymphoid hyperplasia (RH), polymorphic-LPD (Poly-LPD), diffuse large B-cell lymphomas (DLBCLs), and classic Hodgkin lymphoma (CHL), respectively. The clinicopathologic features of RH, Poly-LPD, DLBCLs, and CHL were as follows: extranodal involvement: 13.8% (4/29), 36.4% (12/33), 69.5% (73/105), and 15.4% (4/26); Epstein-Barr virus encoded RNA positivity: 55.2% (16/29), 71.9% (23/32), 45.3% (48/106), and 76.9% (20/26); necrosis: 0% (0/29), 51.5% (17/33), 34.3% (36/105), and 12.0% (3/25); and Hodgkin Reed-Sternberg-like cells: 17.2% (5/29), 50% (14/28), and 19.8% (21/106). The median duration from MTX withdrawal to the time of disease regression was 10.4, 3.0, 4.2, and 2.7 months for RH, Poly-LPD, DLBCLs, and CHL. After MTX withdrawal, progression-free survival was the greatest for RH, followed by for Poly-LPD, DLBCL, and CHL (all P

Tyrosine kinase inhibitor (TKI) treatment is the standard of care for patients with chronic myeloid leukemia (CML). Even in the imatinib era, the presence of 'clonal chromosomal abnormalities' in the Philadelphia chromosome (CCA/Ph+) at diagnosis reportedly increased the risk of disease progression and predicted shorter survival. However, it remains unclear whether CCA/Ph+ is a poor prognostic marker in the era of new‑generation TKIs. The data of patients with CML in the chronic phase (CP) that were extracted from the CML Cooperative Study Group database were retrospectively analyzed. Of the 328 eligible patients, 33 (10.1%) had CCA/Ph+, including 9 major route and 24 minor route aberrations. The characteristics of patients with and without CCA/Ph+ were similar; however, the proportion of blasts was higher among patients with CCA/Ph+. Notably, the survival rate of patients with CCA/Ph+ was not inferior to that of patients without CCA/Ph+, and there were no differences in responses to TKIs. All 9 patients with major route CCA/Ph+ attained a major molecular response (MMR) with no disease progression, and 8 ultimately achieved a deep molecular response. In particular, the median interval between TKI initiation and achievement of MMR was shorter in patients initially treated with a second‑generation TKI than in those treated with imatinib (5 vs. 10 months). The present retrospective study, thus, revealed favorable treatment outcomes in CML‑CP patients with CCA/Ph+ treated with second‑generation TKIs. The data indicated that administering second‑generation TKIs as first‑line treatments is preferable in CML‑CP patients with CCA/Ph+.

Recently, attention has been focused on methotrexate-induced lymphoproliferative disease (MTX-LPD), and atypical phenotypes are occasionally documented. We encountered two patients with rheumatoid arthritis (RA) who were diagnosed with non-specific LPD (LPD-nos). Biopsy samples were not obtained during the initial examination when the LPD development was discovered, and the patients achieved a complete response after MTX cessation (case 1) or steroid pulse therapy (case 2). However, the tumors flared up 1.5 years later, and LPD-nos was determined following biopsies of the lymph node (LN, case 1) and liver (case 2). Prednisolone was subsequently administered instead of chemotherapy; however, multiple masses, including in the spine (case 1), and severe icterus with liver dysfunction (case 2) were exacerbated within a few months. Although the re-biopsy of LN proved the presence of HL and radiation followed by aggressive chemotherapy rescued the patient (case 1), the superficially accessible biopsy site was not found, and autopsy finally revealed HL (case 2). In both cases, the underlying pathogenesis along with the B symptoms and laboratory abnormalities suggested MTX-LPD, HL in particular. Therefore, even if the pathological diagnosis does not confirm the specific LPD subtype, the administration of aggressive chemotherapy should be considered if the LPD activity flares severely.

Thrombopoietin receptor (TPO-R) agonists have been shown to be effective in refractory chronic immune thrombocytopenia (ITP); however, their efficacy in patients under critical care is not known. We report the case of a female patient with a newly diagnosed ITP who experienced severe bleeding from an external wound. The patient was administered the standard treatments for ITP, which are high-dose intravenous immunoglobulin (IVIg) and corticosteroids. However, following failure of these treatments, we administered romiplostim on day 6 after the onset of ITP. On day 6 after the initiation of romiplostim, there was improvement in platelet count and bleeding tendency. We were subsequently able to perform a splenectomy successfully. The efficacy of TPO-R agonists in ITP has been reported in several situations, including before surgery in an ITP patient; however, the use of TPO-R for arterial bleeding with shock has not been reported. To our knowledge, the present article is a rare case report of the use of a TPO-R agonist in a patient with critical artery injury. Our data suggest that the early use of romiplostim is effective in emergency cases of newly diagnosed ITP with life-threatening bleeding, which is refractory to standard treatment.

Background and Aim: Treatment with a tyrosine kinase inhibitor (TKI) is the standard of care for patients with chronic myeloid leukemia (CML). Based on the results of the ENESTnd and DASISION studies, the European LeukemiaNet 2013 guidelines now recommend the use of TKIs, imatinib (400 mg once daily), nilotinib (300 mg twice daily), and dasatinib (100 mg once daily) as first-line treatment for patients with CML in the chronic phase (CML-CP). Compared to imatinib, the new generation TKIs, nilotinib and dasatinib, were found to have deeper and faster treatment response rates with acceptable toxicities. However, a direct comparison between nilotinib and dasatinib has never been reported previously. Our study aims to compare the outcomes and molecular responses achieved following the first-line use of these two agents in patients with CML-CP. Patients and Methods: The database of the CML Cooperative Study Group, which includes patients who were initially diagnosed with CML after the introduction of imatinib (April 2001), was reviewed, and patients who were given nilotinib or dasatinib as first-line therapy were identified. The event-free survival (EFS, defined as the loss of treatment efficacy, disease progression, or any cause of death), and rates of cumulative major molecular response (MMR), and deep molecular response (DMR, defined as the depth of MR 4.5) were compared between the nilotinib and dasatinib groups. Further, the predictive ability of the Sokal, Hasford, and European Treatment and Outcome Study (EUTOS) scoring systems for the achievement of molecular responses was also evaluated. For the analysis of molecular responses, patients who switched from their initial treatment agent to another TKI before achievement of MMR or DMR were considered to have no MMR or DMR. Results and Discussion: Out of 361 patients with CML-CP enrolled in our database, 58 and 63 were treated with conventional doses of nilotinib (300 mg twice daily) and dasatinib (100 mg once daily), respectively, as first-line therapy. Patients who had been started on a low dose TKI therapy were excluded from this analysis. The patient demographics, including age, sex, observation periods, and the Sokal, Hasford, and EUTOS scores did not show significant differences between the groups. In total, there were five events during the observation period (1 in the nilotinib group and 4 in the dasatinib group), and all events were deaths unrelated to CML, except for one in a patient in the dasatinib group who showed loss of complete cytogenetic response. The disease did not progress to the accelerated or blastic phase in any of the cases. The EFS did not differ between these two groups (p = 0.214). The MMR rates by 6, 12, 18, and 24 months were 59%, 72%, 74%, and 81%, respectively, in the nilotinib group and 52%, 73%, 81%, and 86%, respectively, in the dasatinib group. The DMR rates by 6, 12, 18, and 24 months was 7%, 17%, 24%, and 28%, respectively, in the nilotinib group and 3%, 16%, 25%, and 29%, respectively, in the dasatinib group. During the first 24 months of treatment, 4 (7%) patients in the nilotinib group and 11 (17%) in the dasatinib group had been switched to other TKIs (p = 0.0983). Among the three scoring systems, only the Hasford score could predict the achievement of DMR, and all of them failed to predict the achievement of MMR in the entire cohort. Our data suggest that both nilotinib and dasatinib have comparable efficacies, with high molecular response rates and promising outcomes. The validity of our findings should be tested in a randomized study, which is currently underway in Japan. Disclosures Takaku: Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Speakers Bureau. Tokuhira:Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Chugai: Speakers Bureau. Asou:Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Asahi Kasei Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Kizaki:Nippon Shinyaku,: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Speakers Bureau. Kawaguchi:Novartis Pharma K.K.: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Alexion: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

We performed a retrospective study to evaluate the incidence of second malignancies (SMs) in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). We analyzed data from 339 patients with CML who were extracted from the CML Cooperative Study Group database. The standardized incidence ratio (SIR) was calculated to assess the risk of SMs using data from the Cancer Registries in Japan. The median follow-up was 65 months. SMs developed in 14 patients (4.1%, 10 men, 4 women) after the start of TKIs. The median age was 69 years at the time of the CML diagnosis and 72.5 years at the time of the SM diagnosis. Ten patients were treated with imatinib, three with dasatinib, and one with nilotinib as the initial treatment. The SIR for all malignancies was 1.05 (95% CI 0.50–1.93) for men and 1.08 (95% CI 0.29–2.76) for women. The difference in the overall survival (OS) of patients with or without SMs was not statistically significant (5-year OS: 82.5% vs. 92.9%; p = 0.343). A subgroup analysis of 166 patients treated with second-generation TKIs (92 dasatinib, 74 nilotinib) showed that the SIRs for all malignancies were 1.33 (95% CI 0.36–3.41) for men and 0 for women. In conclusion, the incidence of SMs in CML patients during TKI treatment was the same as that in the general Japanese population. There was no evidence of an increase in the incidence of SMs during second-generation TKI treatment. Furthermore, the occurrence of SMs during TKI treatment did not affect the survival or mortality in our cohort.

Background: Lymphoproliferative disorders (LPD), including malignant lymphoma, is a relatively rare but life-threatening complication in RA patients under methotrexate (MTX) therapy. Spontaneous regression of LPD after MTX withdrawal is regarded as a distinct characteristic in part of such LPDs. Objective: The present study aimed to investigate the immunological difference in regressive LPD and persistent LPD. Methods: We studied RA patients who developed LPD during MTX administration (n = 35) and clinically matched controls (n = 35). The time of MTX cessation was defined as week 0, and LPD patients were divided into two groups according to LPD status at week 12: regressive group (n = 22) and persistent group (n = 13). Flow cytometric analysis of whole blood samples and serum cytokine assays were conducted for LPD (n = 10) and control patients (n = 10) at weeks 0, 4 and 12. Results: There was a significant decrease in peripheral lymphocytes and the proportion of T helper 1 cells (Th1 cells), effector memory CD8+ T cells (EMCD8+ T) and Epstein–Barr virus (EBV)-specific CD8+ T cells at the time of LPD diagnosis, and a significant increase after MTX cessation was observed in the regressive group but not in the persistent group. The expansion of Th1 cells and EMCD8+ T cells significantly correlated with an increase in serum interferon (IFN)-γ concentration. Conclusion: Changes in Th1 cells, EMCD8+ T cells and EBV-specific CD8+ T cells, which coincided with an increase in IFN-γ, were significantly different between regressive LPD and persistent LPD after MTX cessation.

This study investigated the incidence rate and features of vascular adverse events (VAEs) in Japanese patients with chronic myeloid leukemia (CML) who were treated with tyrosine kinase inhibitors (TKIs). The analysis included 369 CML patients in the chronic or accelerated phases, selected from the CML Cooperative Study Group database; 25 events in 23 (6.2%) of these patients were VAEs. At the time of VAE incidence, nine patients were on treatment with imatinib, 12 with nilotinib, three with dasatinib, and one with bosutinib. VAE incidence comprised 13 cases of ischemic heart disease (IHD), eight of cerebral infarction (CI), and four of peripheral arterial occlusive disease (PAOD). IHD incidence rate in the study population was higher than that in the age-matched general population, particularly in nilotinib-treated patients, while CI incidence rate was almost equivalent. Compared with the Suita score, the SCORE chart and the Framingham score risk assessment tools detected more patients with high or very high risk of VAEs. In conclusion, incidence of IHD requires closer monitoring in nilotinib-treated patients. More detailed investigations for determining the most useful tool to predict VAE incidence and long-term analysis of therapy-related VAE cases are needed for improving safety during TKI therapy.

The purpose of this clinical trial was to evaluate the efficacy of 2-year consolidation therapy using nilotinib (NIL) for achieving a molecular response (MR4.5, BCR-ABL1IS ≤ 0.0032% on the International Scale) in patients with chronic myeloid leukemia in the chronic phase (CML-CP) who had achieved a major molecular response (MMR, BCR-ABL1IS ≤ 0.1%) with imatinib (IM). We recruited 76 Japanese patients for this trial. Nilotinib 300 mg, twice daily, was administered for 2 years, and 74 patients were evaluated in the study. The median age was 55.0 years. The median duration of IM treatment was 69.0 months. All patients showed MMR at the time of entry into the study; the median time to MMR on IM therapy was 20.4 months. The proportion of patients who achieved MR4.5 increased over time. The rates of MR4.5 in the 74 evaluable patients were 27.0% [90% confidence interval (CI) (18.7–36.8%)] and 44.6% [90% CI (34.7–54.8%)] at 12 and 24 months, respectively. The frequency of ABCG2 421C/A + A/A was an independent predictive biomarker for achieving a 24-month MR4.5. Switching to NIL led to safer, deeper molecular responses in patients with MMR on long-term IM therapy for future treatment-free remission.

ABL1-tyrosine kinase inhibitors (TKIs) have led to dramatic changes in treatment strategies for chronic myeloid leukemia in the chronic phase (CML-CP). However, clinical studies have highlighted increasing numbers of adverse events (AE) with TKIs. Although TKI modification plays a key role in AE management, this process is poorly understood, particularly in terms of the TKI nilotinib. In the present study, we retrospectively analyzed the records of 70 patients with newly diagnosed (ND)-CML-CP who were treated with nilotinib to investigate the drug potency of nilotinib and treatment management. During a median observation period of 3.4 years, 76% of patients continued nilotinib as a first-line treatment. The 1-year and overall major molecular response (MMR) rates and the overall molecular response (MR) 4.5 rate for all patients receiving first-line nilotinib therapy were 70, 84.2, and 50%, respectively. No case progressed to the accelerated or blast phase during the study. To avoid AEs during the early phase, nilotinib doses were reduced to

Although recent accumulative data reveal the clinicopathogenesis of regression in methotrexate-induced lymphoproliferative disorders (MTX-LPDs), the precise understanding including this category remains controversial. In this study, we analyzed 62 patients with MTX-LPD. Forty-three patients showed regression (Reg group), with high rates of Hodgkin lymphoma (HL) and LPD (90 and 88%, respectively). Among the 43 patients of the Reg group, 14 patients (33%) relapsed. The median duration before relapse in the Reg group was 10.6 months. Although the difference of OS between the Reg and Non-Reg groups was not significantly different, relapse-free patients in the Reg group had a superior overall survival (OS). MTX duration had a significant impact on Epstein-Barr virus (EBV) infection (p = .00131). Furthermore, EBV infection was significantly related to clinical manifestations, including spleen invasion, in the regression phenomenon. Some human leukocyte antigens (HLA) alleles might affect MTX-LPD development via EBV infection, although A*2402 and DRB1*0405 might be affected as fundamental factors.

Background Lympho-proliferative disorder (LPD) is known as a relatively rare but life-threatening complication in RA patients under MTX administration. Spontaneous regression of LPD after MTX withdrawal is regarded as a distinct character of LPD under MTX administration. Previous study from our institution [1] and others [2] reported the link between decreased lymphocyte counts at LPD diagnosis and restoration after MTX cessation and the regression of LPD. Objectives To investigate the immunological factors including lymphocyte subsets which involved in spontaneous regression of LPD following MTX withdrawal. Methods We studied 35 RA patients complicated with LPD under MTX administration in our institution. Age, sex, RA disease duration matched control MTX-treated patients (N=35) were selected. LPD patients were divided into two groups regarding to the status of LPD after MTX cessation; regressive group (N=22) and persistent group (N=13). Clinical features were compared among 3 groups. Flowcytometric analysis of the whole blood sample and measurement of cytokine concentration in ELISA were conducted in a part of the LPD patients (N=10, 7 regressive, and 3 persistent LPD) and controls (N=10). The time of MTX cessation, which was simultaneous with LPD diagnosis, was defined as week 0, and blood sample was collected at week 0, 4 and 12. Results At week 0, number of peripheral lymphocytes was significantly decreased in regressive group, compared to persistent group and control group. Flowcytometric analysis revealed significant decrease in proportion of effector memory CD8+ T cells (EMCD8+T), Epstein Barr Virus specific CD8+ T cells (EBV specific CD8+) and T helper 1 cells (Th1 cells) subset in regressive group compared to control group. Following MTX cessation, significant increase in proportion and absolute number of these subsets were observed only in the regressive group, but not in persistent group. Expansion of Th1 cells and EMCD8+ T cells significantly correlated with increase of serum IFN-γ, and expansion of EMCD8+ T cells inversely correlated with change of serum IL-15. Conclusions Restoration in proportion and absolute number of Th1 cells, EMCD8+T cells and EBV specific CD8+ T cells coincided with increase of IFN-γ, and associated with regression of LPD developed under MTX administration. Since changes of those immunological factors were not observed in persistent LPD, this study would be the first report to demonstrate the difference of immune status between regressive and persistent LPDs developed under MTX administration. References Saito S, et al. Rheumatology. 2017; in press. Inui Y, et al. Leuk Lymphoma. 2015; 56(11):3045–3051. Acknowledgements None. Disclosure of Interest S. Saito: None declared, K. Suzuki: None declared, K. Yamaoka Speakers bureau: Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma, Takeda Industrial Pharma, GlaxoSmithKline, Nippon Shinyaku, Eli Lilly, Janssen Pharma, Eisai Pharma, Astellas Pharma and Actelion Pharmaceuticals, K. Amano: None declared, M. Tokuhira Speakers bureau: Eisai Co., T. Takeuchi Grant/research support from: Astellas Pharma, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Eisai Co.,Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co.,Ltd., Teijin Pharma Ltd., AbbVie GK, 2,Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K.

The development of novel therapeutic agents over the past decade, including the proteasome inhibitor, bortezomib, and the immunomodulatory drugs, lenalidomide and thalidomide, has resulted in improved outcomes for patients with multiple myeloma. However, there is still considerable controversy as to which regimen should be used as first-line therapy, which patients should be considered for autologous or allogeneic transplantation, and how consolidation or maintenance therapy is used in patients that have a good response to first-line therapy. The present paper will review clinical evidence from previous and ongoing studies to explore issues related to these questions.

Objective To clarify the characteristics of lymphoproliferative disorder (LPD) in patients with RA treated with MTX. Methods Among 33 patients developing LPD during MTX treatment, 20 LPDs regressed spontaneously within 12 weeks after MTX cessation (regressive LPD), and 13 did not regress and most of them died or needed chemotherapy (persistent LPD). The control group consisted of 66 clinically matched MTX-treated RA patients without LPD. The clinical characteristics were compared between these three groups. Results While no significant differences were found in clinical RA and LPD features among the three groups, the absolute lymphocyte number of the two LPD groups at LPD diagnosis was significantly lower than the control group (497/µl in the regressive vs 680/µl in the persistent vs 1400/µl in the control, P < 0.05). After MTX withdrawal, the lymphocyte number in the regressive group rapidly recovered to 1214/µl (P < 0.01) by week 2 and was thereafter maintained at an equivalent level to the control group. In contrast, lymphocyte level in the persistent group did not show significant increase throughout 12 weeks (620/µl at week 2, P = 0.57). Changes in lymphocyte number following MTX withdrawal clearly distinguished the regressive LPD from the persistent LPD. Conclusion A significant decrease in lymphocyte count at the LPD diagnosis and its restoration after MTX withdrawal were markedly associated with spontaneous regression of LPD developing during MTX treatment.

Background: According to VISTA study, the prognosis of elderly multiple myeloma (MM) patients has been improved with VMP therapy. However, intensive chemotherapy has induced severe adverse events (AEs), resulting in highly discontinued rate, especially often seen in unfit or frail patients. Several studies have demonstrated the potent of weekly bortezomib administration in induction therapy (GIMEMA, GEM2005MAS65), and maintenance therapy with bortezomib. Based on these fact, we designed phase 2 clinical study, BoRtezomib-based Optimized therapy Aiming Disease control in Japan (BROAD-J study), based on a weekly VMP as induction therapy followed by maintenance therapy with bortezomib (Bor-MT) with every two weeks administration for newly-diagnosed (ND) symptomatic non-transplant eligible MM patients. (UMIN 00007335) Method: From August 2011 to June 2016, according to the International Myeloma Working Group (IMWG) criteria, 87 patients older than 65 years with symptomatic MM were eligible for this trial (Table 1). The primary objectives were included maximum response and time to progression duration. Secondary objective was the continued treatment duration and adverse event rate. The induction phase consisted of VMP treatment: Melpharan: on days 1-4 (6 mg/m2), every 35-day cycle; prednisolone: on days 1-4 (40mg/d, every 35-day cycle; Bortezomib: 1.3mg/m2, d1, 8, 15, 22 every 35-day cycle. After 9 cycles of VMP therapy, the maintenance therapy starts. The maintenance phase was consisted of twice a month bortezomib administration (1.3 mg/m2) without dexamethasone until progressive disease. Dose reductions of bortezomib were allowed according to instructorfs recommendation. Patients could receive supportive therapy including bisphosphonates and transfusions as necessary. AEs were graded according to NCI-CTCAE v4.0. Response was assessed prior to every treatment cycle. Response categories were based on the International Myeloma Working Group uniform response criteria. Results: Mean age was 75 years (66-88), sex ratio was 44:43 (M: F), and ISS stage was I 12%, II 43%, III 43%. High risk cytogenetics abnormalities (i.e. del17p, t(14;16)) were observed in 13 patients (15%). Overall response rate was 87% and complete response was obtained in 22 patients (25%) (Table 1). Median progression free survival (PFS) was 36 months (Figure 1a) and median overall survival was 57 months (Figure 1b). Median PFS (Kaplan-Meier estimate) was significantly higher with patients who obtained CR or VGPR than PR or SD (P Discussion: This trial reveals that VMP regimen with weekly bortezomib administration is effective and tolerablefor unfit or frail elderly MM patients. In addition, maintenance therapy of twice a month bortezomib administration was effective in patients who obtained VGPR or more better. These results are no way inferior to results of VISTA trial. However, high R-ISS stage patients or poor treatment responders did not obtain the benefit of this setting, so, another setting with anti-myeloma antibody (i.e. elotuzumab or daratumumab) may be warranted in the future for such patients. Conclusion: BROAD-J study is conducted to yield the possibility of a careful treatment toward at least PR state and the efficacy of VMP followed by bortezomib maintenance in unfit or frail MM patients. The excellent results we demonstrated such as a good OS, PFS, and a low incidence of AEs might be suggested a superiority of this strategy. Download : Download high-res image (64KB) Download : Download full-size image Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria.

Introduction: Deferasirox is an oral iron chelator, approved worldwide for the treatment of chronic iron overload due to transfusion. Deferasirox was permitted two years ago in Japan, but there is little known regarding its efficacy and tolerability in clinical practice. Methods: We conducted a retrospective study of 18 patients with transfusion-dependent anemias treated by deferasirox at our institution. The starting dose was individualized and ranged from 6.4 to 26.3 mg/kg/day. Routine clinical laboratory data were followed, and serum ferritin was assessed every 4 weeks. Results: The mean serum ferritin level of 18 patients at the time of deferasirox induction was 3162 ng/ml. Ten of 18 patients could sustain deferasirox treatment for at least 6 months, at an average maintenance dose of 10.8 mg/kg/day. Serum ferritin reduction was observed in 4 patients, at doses less than 20 mg/kg/day. Eighty-nine percent of the patients had adverse events, and 13 of them in all ultimately discontinued. Myelodysplastic syndrome (MDS) patients showed poor tolerability. Severe infections of grade 3 or more were documented in 6 patients, and 2 of them were fatal. Conclusions: The potential for beneficial iron chelation of deferasirox at less than the recommended 20 mg/kg dose was demonstrated. On the contrary, poor tolerability was documented, with adverse events such as severe infections, especially in MDS patients. Although it was not clearly demonstrated that deferasirox was responsible for impaired immunity, careful watching is required to administrate deferasirox.

[Background] Methotrexate - induced lymphoproliferative disorders (MTX-LPDs) belong to the group of other iatrogenic immunodeficiency-associated LPDs, as defined in the revised 4th edition of the World Health Organization (WHO) classification. The phenomenon of LPD regression after MTX withdrawal is a specific event that develops in patients with autoimmune diseases (ADs), such as rheumatoid arthritis (RA). Although patients with regressive MTX-LPDs have better overall survival (OS), relapse/regrowth (R/R) events after regression are one of the factors of poor prognosis. Recent studies including ours have shown that lymphocytes play an important role in the regressive LPD phenomenon in MTX-LPDs (Rheumatology (Oxford). 2017;56:940-946, Front Immunol. 2018;4;9:621) ; however, little is known of the details regarding differences among the LPD subtypes and the influence of lymphocytes on R/R events. In this study, we analyzed patients with regressive Hodgkin lymphoma (HL) and diffuse large B cell lymphoma (DLBCL), especially focusing on the influence of the absolute lymphocyte count (ALC) on R/R events and clinical outcomes. [Methods] Data were collected from 25 patients with ADs who developed LPD and had regressive LPD after MTX withdrawal at our institutions. All diagnoses were confirmed based on immunohistochemistry analysis of paraffin-embedded samples. All pathological samples were classified and diagnosed according to the WHO classification. The ALCs were determined at the time of MTX withdrawal due to LPD development (0 M), 1 month after MTX withdrawal (1 M), 6 months after MTX withdrawal (6 M), and at the time of R/R. The OS was calculated between 0 M and the time of the last observation. Statistical analyses were performed using EZR software. [Results] Of 25 patients with regressive MTX-LPDs, the median age at 0 M was 67 years (range: 44ー84). Ten men and 15 women were included. The basal ADs were RA (N = 23), psoriasis vulgaris (N = 1), and systemic lupus erythematosus (N = 1). The median duration of MTX treatment was 5.5 years. Seventeen cases of DLBCL and 8 cases of HL were included. The median ALCs at 0 M and 1 M in all patients were 627/µL and 1364/µL, respectively. The median ALC ratio (ALC at 1 M divided by ALC at 0 M) was 2.3 (0.6-8), and it indicated over 1 in 24 patients, confirming ALC recovery at 1 M. Regarding the ALCs at 0 M and 1 M, significant differences between DLBCL and HL were not detected (p=0.215 and p=0.77, respectively). Of all patients, the median duration from the time of MTX duration to R/R was 12 months (range, 7-41). Thirteen patients remained in the remission state without R/R (Regression group), whereas 12 patients experienced an R/R event (R/R group). In the latter group, the median ALCs at 0 M, 1 M, 6 M, and R/R were 582/µL, 1194/µL, 1158/µL, and 565/µL, respectively. The ALC significantly increased at 1 M from 0 M (p=0.006) and decreased at R/R from 1 M (p=0.05), suggesting that the ALC recovery diminished when the LPD underwent R/R. On the other hand, among 13 patients in the regressive group, the median ALCs at 1 M, 6 M, and at the time of the last observation were over 1500/µL (1732/µL, 1782/µL, and 1574/µL, respectively), although that at 0 M was less than 1000/µL (991/µL). To investigate the influence of ALC on the OS, we analyzed the clinical definition of the ALC cut-off of 1000/µL after the ALC recovery observed at 1 M. The result indicated that patients with ALC greater than or equal to 1000/µL after 6 M had significantly better OS compared to those with ALC less than 1000/µL after 6 M (5-year OS, 100% vs. 43.8%, p=0.00048), considering that the ALC is one of the strong prognostic factors in the clinical outcomes of MTX-LPDs. It is of note that the R/R rate in patients with HL was higher than that in those with DLBCL (100% and 23.4%, respectively), and all 8 patients with HL had an ALC at R/R of less than 1000/µL. [Summary] In this study, we demonstrated that ALC recovery was detected at 1 M, which continued during the clinical course in patients who maintained a remission state, although it decreased to less than 1000/µL after ALC recovery in patients with R/R LPDs. In addition, the ALC of 1000/µL was a critical level affecting the clinical outcomes. Regarding LPD subtypes, the reason for higher R/R rates observed in HL compared to those in DLBCL was thought to be dependent on the rates of lower ALCs. [Conclusion] Our data suggest that the ALC is one of the predictive factors for R/R and OS in patients with regressive MTX-LPDs. Disclosures Tokuhira: Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Chugai: Speakers Bureau. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau. Kizaki:Nippon Shinyaku,: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Speakers Bureau.

The expression of YB-1 has been reported to predict poor clinical outcome in many human malignancies, including hematopoietic malignancies. In this study, we investigated the correlations between YB-1 expression and the clinicopathological features of patients with diffuse large B-cell lymphoma (DLBCL) in a single institution. The expression of YB-1 was analyzed in 168 cases by immunohistochemistry. Fifteen out of 168 cases (8.9%) showed cytoplasmic expression of YB-1. The expression of YB-1 was significantly associated with 5-year overall survival (OS) (p = 0.023). Rituximab plus CHOP therapy (n = 94) improved the 5-year survival rate in both YB-1-positive and -negative patients. In conclusion, the data presented in this report provide evidence that the cytoplasmic expression of YB-1 is a poor prognosis factor in DLBCL treated with CHOP therapy, whereas rituximab improves the survival of both YB-1-positive and -negative patients with DLBCL.

Although the introduction of novel agents, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors (HDACi) has improved the clinical outcomes of the patients with multiple myeloma (MM), most patients eventually relapse and develop drug resistance. Proteasome inhibitor, bortezomib (BTZ), is a key drug for the treatment of MM, and the prognosis of the patients with BTZ resistance is very poor. Therefore, novel therapeutic approaches to overcome BTZ resistance are urgently needed. WEE1 is a cell cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 induces cell cycle arrest, allowing for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells from mitotic lethality. WEE1 is overexpressed in various cancers, and relates to poor prognosis. In addition, the inhibition of WEE1 has been shown to induce cancer cells to premature mitotic entry leading to apoptotic cell death, suggesting that combining WEE1 inhibitor with DNA damaging agent is effective in cancer therapy. Resent research has shown that HDACi increase reactive oxygen species production resulting in increased DNA damage and impaired DNA repair. These data suggest that the combination of WEE1 inhibitor and HDACi might be an attractive therapeutic option against this incurable hematological malignancy. In the present study, we investigated the efficacy of WEE1 inhibitor MK-1775, a potent and highly-selective small molecule inhibitor of WEE1, in combination with HDACi in various MM cells. MTS assays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including BTZ-resistant cell line, in a dose- (0 to 10 μM) and time- (0 to 72 h) dependent manner. In combination with vorinostat which is a pan-HDACi, MK-1775 inhibited the proliferation of BTZ-resistant MM cells more effectively than single agent. Furthermore, the combination of vorinostat and MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. Assays for apoptotic cell death demonstrated that MK-1775 induces both early and late apoptosis in MM cells. Western blotting analysis was performed to investigate the molecular mechanisms of MK-1775-induced cell death, showing that MK-1775 inhibited the expression of the target molecules of Bcl-2 and MCL1, and CDK1 phosphorylation. Moreover, the marked increase in PARP and Caspase 3 cleavage, and histone H2AX expression was observed following treatment with MK-1775 in combination with vorinostat. These findings suggest that the combination of MK-1775 and vorinostat induces synergistically DNA damage and promotes premature mitotic entry resulting in apoptotic cell death in MM cells. In conclusion, our data suggest that WEE1 in combination with HDACi might be a promising molecular target for the treatment of BTZ-resistant MM. Citation Format: Takayuki Tabayashi, Yuka Tanaka, Yasuyuki Takahashi, Yuta Kimura, Tatsuki Tomikawa, Tomoe Anan, Morihiko Sagawa, Reiko Watanabe, Michihide Tokuhira, Masahiro Kizaki. Dual targeting of cell cycle checkpoint and histone deacetylase overcomes bortezomib resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1884.

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial β-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1β by 85% after 24 h. Likewise, TNF-α levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-α levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1α production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.

SummaryHemostatic molecular markers were serially monitored in a prospective fashion during remission induction therapy with all-trans retinoic acid (ATRA) in sixteen patients with acute promyelocytic leukemia (APL). One patient with leukocytosis before treatment and three patients who later developed hyperleukocytosis also received chemotherapy with behenoyl Ara-C and daunorubicin. Plasma levels of E-fragment of fibrin and fibrinogen degradation product (FDP-E), FDP-D dimer (D-D), thrombin-antithrombin complex (TAT), and plasmin-α2 plasmin inhibitor complex (PIC) were markedly elevated in all but one patient before treatment, and these parameters decreased to normal or near normal ranges in most patients within the first 7 days of treatment. Interestingly, we have found that these parameters were again elevated during the later course of ATRA therapy (after day +7) in eleven patients for various reasons including cytotoxic chemotherapy (3 cases), fever (5 cases; 2 cases with apparent infection, 3 cases without known etiology), Caesarean section (1 case), and no apparent etiology (2 cases). Three patients showed bleeding complications during reelevation of molecular markers, but none developed thrombosis. Plasma elastase-α1 proteinase inhibitor complex (E-α1PI) was markedly elevated in all patients at diagnosis and did not decrease significantly during ATRA therapy. Plasma tissue factor antigen was mildly elevated in one out of four patients studied, and thrombomodulin was elevated in two out of ten patients tested. These results confirmed the rapid normalization of coagulopathy during the early phase of remission induction therapy with ATRA but suggest that re-elevation of molecular markers occurs frequently during the later course of ATRA therapy.

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

SummaryWe characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the αIIbβ3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37°C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody’s epitope depends upon a conformational structure maintained by αβ subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the αIIbβ3 integrin on unstimulated, quiescent platelets.

Multiple myeloma (MM) is a hematological malignancy that derives from the proliferation of unregulated plasma cells. Dramatic improvement in the clinical outcomes of both newly diagnosed and relapsed/refractory patients with MM has been achieved using many clinical approaches, including use of high-dose chemotherapy followed by hematopoietic stem cell transplantation, and new drugs, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors. However, most patients eventually relapse and develop drug resistance. Moreover, the prognosis of patients with bortezomib (BTZ) and/or lenalidomide (LEN)-resistant MM (key drugs in the treatment of MM) is very poor. Therefore, novel therapeutic approaches to overcome BTZ and LEN resistance are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 catalyzes inhibitory phosphorylation of both cyclin-dependent kinase1 and 2 (CDK1 and CDK2), leading to CDK1- and CDK2-induced cell cycle arrest at the G1, S, or G2-M phases. This cell-cycle arrest, in turn, allows for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells harboring unrepaired damaged DNA from mitotic lethality. Furthermore, recent research has shown that knockdown of WEE1 leads to DNA double-strand breaks specifically in S-phase cells undergoing DNA replication, and that WEE1 is most active in the S-phase, suggesting that WEE1 is involved in DNA synthesis. Overexpression of WEE1 has been observed in many types of cancers, including hepatic cancer, breast cancer, glioblastoma and gastric cancer, and high expression of WEE1 has been shown to correlate with poor prognosis. In addition, research has shown that inhibition of checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response, potentiates the cytotoxicity of chemotherapy on p53-deficient MM cells, which are regarded as chemotherapy-resistant, suggesting that inhibition of cell-cycle checkpoint kinase is involved in re-sensitization of refractory MM cells to anticancer drugs. These data suggest that WEE1 might be an attractive target for novel therapeutic agents against this incurable hematological malignancy. MK-1775 is a potent and highly-selective small-molecule inhibitor of WEE1. In the present study, we investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775. MTSassays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including the intrinsically LEN-resistant cell line, RPMI-8226, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. Furthermore, the growth inhibition effect is irrespective of p53 status. To examine the mechanisms behind the growth inhibition effect induced by MK-1775, assays for apoptotic cell death were performed. These assays demonstrated that MK-1775 induces both early and late apoptosis in MM cells. To investigate the molecular mechanisms of MK-1775-induced cell death in MM cells, the expression of various cell death-associated proteins and downstream molecules of WEE1 were examined. Western blotting analysis showed that MK-1775 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by inhibiting both, the expression of the target molecules of Bcl-2 and MCL1, and the cleavage of PARP and Caspase 3. Similarly, there was a substantial inhibition of CDK1 phosphorylation downstream of WEE1. Moreover, an increased expression of histone H2AX was observed following administration of MK-1775, suggesting that MK-1775 results in cytotoxicity by direct DNA damage. Next, we examined the effects of MK-1775 on BTZ-resistant MM cells. Interestingly, MK-1775 inhibited the proliferation of both BTZ-sensitive wild-type MM cells and BTZ-resistant MM cells, suggesting that BTZ resistance can be overcome by targeting WEE1. Furthermore, in combination with BTZ, MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that inhibition of WEE1 might serve as an attractive therapeutic option for patients with both BTZ-resistant and LEN-resistant MM. In conclusion, our data suggest that WEE1 might be a promising molecular target for the treatment of MM. Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria.

【Background】Despite numerous attempts to elucidate the mechanism underlying other iatrogenic immunodeficiency-associated lymphoproliferative diseases (OIIA-LPDs), the mechanism remains poorly understood. Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is one of the disease entities included in OIIA-LPDs, which is especially developed in rheumatoid arthritis (RA) as we demonstrated (Tokuhira et al, Leuk Lymphoma. 2012;53:616-23). Spontaneous regression of LPD after MTX withdrawal is one of the distinct characteristics of MTX-LPD, and recent articles suggest that the increase in peripheral blood (PB) lymphocytes affect this phenomenon. However, the population of lymphocytes necessary to induce MTX-LPD regression is still unknown. 【Methods】We prospectively analyzed 10 patients with RA developing confirmed LPD during MTX administration, in our institutions between January 2014 to October 2015. All patients ceased MTX after developing lymphoadenopathies. To investigate the factors involved in spontaneous regression of LPD following MTX withdrawal, we performed flowcytometric analysis of whole blood including lymphocytes from PB for CD3, 4, 8, 14, 15, 20, 56, 127, and 45RO, HLA-DR, CCR3, CCR4, CCR6, CCR7, and EBV-tetramer expression, for all patients at three time points during the clinical course of treatment; at the time of MTX cessation (w0), and 4 weeks (w4) and 12 weeks (w12) after MTX cessation. We selected 10 age-, sex-, MTX dose-, and RA duration-matched RA patients treated with MTX for more than 6 months as controls, randomly selected from among the RA patients at our institutions. The patients with MTX-LPD were divided into two groups based on the status of LPD after MTX cessation, regressive LPD (R group) and persistent LPD (P group), and clinical parameters were compared. 【Results】In the 10 RA patients with MTX-LPD, the median age was 62.4 years (40-74), and the ratio of male:female patients was 2:8. The median duration from the time of RA diagnosis to the time of MTX development was 7.2 years (1.2-20.4), and the median duration of MTX administration was 4.3 years (0.7-9.8). The pathological diagnosis of LPDs was diffuse large B cell lymphoma (DLBCL, n=5), Classical Hodgkin lymphoma (cHL, n = 3), and non-specific LPD (LPD-nos, n=2). The averages values for LDH, CRP, and sIL-2R in the serum were 220 IU/mL (176-554), 0.85 mg/dL (0.06-2.53), and 579 IU/L(206-1210), respectively. The number of patients in the R group and the P group was 7 and 3, respectively. According to LPD phenotypes, all 5 cases of DLBCL belonged to the R group, whereas 1 of 3 HL and 1 of 2 LPD-nos belonged to the R group. At w0, the median WBC count and absolute lymphocyte number in 10 MTX-LPD patients were 5810/µL (2700-11400) and 964/µL (220-2070), respectively. On the other hand, the median WBC count and absolute lymphocyte number in the 10 control patients were 5930/µL (3900-9000) and 1512/µL (755-2379), respectively. The absolute lymphocyte count in the P group was significantly higher than that in the R group. In addition, a significant increase in the lymphocyte number following MTX withdrawal was observed only in the R group, but not in the P group. There were several observations from the flowcytometric analysis. First, the proportions of effector memory CD8+ T cells (EM CD8+), EBV specific CD8+ T cells, and T helper 1 (Th1) cells were significantly decreased both in the R group and in the P group compared to the control group at w0, and this subset was significantly increased in the R group at w4 and w12 compared to the P group. Second, the proportion of CD14-CD15+ cells in the lymphocyte fraction, which includes myeloid-derived suppressor cells (MDSC), was significantly increased in LPD group at w0, and significantly decreased following MTX cessation only in the R group, but not in the P group. Third, the proportion of CD14-CD15+ cells negatively and significantly correlated with the proportion of EM CD8+T cells (r2 = 0.47, p < 0.0001). 【Conclusions】Although the mechanism of MTX-LPD regression following MTX withdrawal is still unknown, this study demonstrates that reconstitution with specific lymphocytes in the PB is definitely correlated with LPD-regression. The proportion of Th1 cells, EM CD 8+, EBV specific CD8+ along with CD14-CD15+ cells dramatically indicated restoration and transition following MTX cessation, suggesting their potent effect toward the progression and regression of MTX-LPD. Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria. Okamoto:Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Shionogi & Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Background Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is a relatively rare but well known complication among patients under MTX treatment. Spontaneous regression of LPD after MTX withdrawal is regarded as a distinct character of MTX-LPD. Although detection of Epstein-Barr virus encoded small RNAs (EBER) in the pathological tissue is reported as a predictor of regression, although it is not necessarily specific 1) . Objectives To investigate the factors involved in spontaneous regression of LPD following MTX withdrawal. Methods We retrospectively reviewed 43 rheumatoid arthritis (RA) patients complicated with MTX-LPD from January 2000 to October 2015 in our institution. Age, sex, RA duration matched 76 control MTX-treated patients were selected. MTX-LPD patients were divided into two groups regarding to the status of LPD after MTX cessation; regressive LPD (R group) and persistent LPD (P group), and clinical parameters were compared. Flowcytometric analysis of the whole blood was underwent in a part of the MTX-LPD patients (N=10, 7 regressive, and 3 persistent LPD) and controls (N=10). The time of MTX cessation (= time of LPD diagnosis) was defined as week 0, and whole blood sample was collected at week 0, 4 and 12. Epstein Barr Virus antigen specific CD8+ T cells (EBV specific CD8+) was defined as MHC/EBV peptide tetramer positive CD8+ T cells. Results At the time of MTX cessation (week 0), number of peripheral lymphocytes was significantly decreased in LPD group, compared to control group. Among the LPD group, significant increase of lymphocyte number following MTX withdrawal was observed only in the R group, but not in P group while MTX dose, EBER positivity rate and pathological phenotypes did not show significant difference. Flowcytometric analysis revealed significant decrease in proportion of effector memory CD8+ T cells (EM CD8+), EBV specific CD8+ and T helpler 1 (Th1) subset in R group compared to control group. Following MTX cessation, significant increase in proportion of these subsets were observed only in the R group, but not in P group. On the other hand, proportion of CD14-CD15+ cells in lymphocyte fraction, which include myeloid derived suppressor cells 2) (MDSC) was significantly increased in LPD group at week 0, and significantly decreased following MTX cessation only in the R group, but not in P group. Proportion of CD14-CD15+ cells negatively and significantly correlated with the proportion of EM CD8+T cells. Conclusions Our study suggested that decreased lymphocytes at the time of LPD diagnosis and restoration following MTX withdrawal are associated with pathogenesis and clinical course of “regressive” MTX-LPD. Proportion of Th1 cells, EM CD 8+, EBV specific CD8+ and CD14-CD15+ cells was restored following MTX cessation, indicating their involvement in regression of MTX-LPD. References Ichikawa A, et al. Eur J Haematol 2013;91:20–8. Austin D, etal. Cancer Immunol Immunother 2013;62:299–307. Acknowledgement none Disclosure of Interest S. Saito: None declared, K. Suzuki Grant/research support from: Eisai Co.,Ltd., Bristol–Myers K.K., K. Yamaoka Paid instructor for: Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma, Takeda Industrial Pharma, GlaxoSmithKline, Nippon Shinyaku, Eli Lilly, Janssen Pharma, Eisai Pharma, Astellas Pharma and Actelion Pharmaceuticals, T. Shimizu: None declared, T. Mori Paid instructor for: Astellas Pharma,Chugai Pharma,Eisai Co.,Ltd.,Janssen Pharmaceutical K.K., S. Okamoto Grant/research support from: Astellas Pharma,Chugai Pharma,Eisai Co.,Ltd.,Bristol–Myers K.K., Takeda Pharmaceutical Co.,Pfizer, Paid instructor for: Astellas Pharma,Chugai Pharma,Eisai Co.,Ltd.,Bristol–Myers K.K.,Takeda Pharmaceutical Co., Pfizer, K. Amano: None declared, M. Tokuhira: None declared, T. Takeuchi Grant/research support from: Astellas Pharma, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Eisai Co.,Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co.,Ltd., Teijin Pharma Ltd., AbbVie GK, 2,Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Paid instructor for: Astellas Pharma, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Eisai Co.,Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co.,Ltd., Teijin Pharma Ltd., AbbVie GK, 2,Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K.

Despite numerous attempts to uncover the mechanism of other iatrogenic immunodeficiency-associated lymphoproliferative diseases (OIIA-LPDs), this mechanism remains poorly understood, especially in rheumatoid arthritis (RA) patients. We analyzed the data on 23 patients with LPDs and RA. Patients were categorized into three groups according to whether they had methotrexate (MTX); MTX-regressive LPDs, MTX-persistent LPDs or other drugs-mediated LPDs. The LPDs seen in OIIA-LPDs-RA might have a unique behavior to think about several rare phenotypes. The overall survival of all patients was 74% at 5 years, and those of the three groups were 100%, 64% and 60%, respectively. Among the 6 patients who died, 4 had LPDs that were detected late, and thus adequate treatment was not given. In addition, several patients with diffuse large B cell lymphoma with a complex karyotype achieved complete remission (CR). Only one among the 17 patients who achieved CR relapsed. OIIA-LPDs-RA appeared to have a better prognosis than other more common types of lymphomas. Regarding RA treatment, various anti-RA drugs were given to the patients after developing LPDs, including MTX, but no recurrent patients were documented.

Bortezomib is a potent agent for multiple myeloma (MM); however, severe treatment-related toxicities such as peripheral neuropathy have been observed in conjunction with its use. In this study, we present the cases of 9 patients with refractory MM whose administration schedule was modified from twice weekly to an interval of once weekly or longer mainly due to adverse events. The average duration from diagnosis to the time of bortezomib induction was 56 months. The schedule was changed to the modified administration according to the physician's discretion. The average duration of modified treatment was 16 months. Six patients with IgG or IgA subtype showed more than a minor response. One patient with BJP had stable disease for 3 years, and the other BJP-type patient with extramedullary plasmacytomas showed remarkable tumor regression. The treatment-related toxicities of this strategy were mild and tolerable. To our knowledge, this is the first report of the administration of bortezomib at intervals longer than once weekly.

Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

Background: Substantial efficacy has been demonstrated with bortezomib (Bor)-containing regimens for the treatment of refractory/relapsed (RR) patients with transplant-ineligible (TI) multiple myeloma (MM). However, the dosing schedules for induction therapy and the significance of maintenance therapy using Bor in clinical settings have yet to be standardized. Discontinuations and dose reduction of Bor owing to severe adverse events (AEs) such as peripheral neuropathy (PN) and infection are frequent causes of adjustments to the standard 9 cycles of Bor/dexamethasone (BD) therapy used for induction in clinical practice. We have previously demonstrated that a change in Bor administration to a schedule of weekly or longer intervals had similar or greater efficacy for patients with RR-MM including transplant-eligible (Tokuhira M, et al. Leuk Res. 35:591-7, 2011). To investigate the efficacy of this modified Bor dosing schedule in the treatment focusing of TI-RR-MM, we retrospectively analyzed 22 patients receiving weekly-BD induction followed by a modified schedule of biweekly or longer intervals using Bor as maintenance therapy in a single institution. Methods: Data on the 22 TI-RR-MM patients treated with BD therapy in our institution were retrospectively analyzed. The induction therapy consisted of intravenous injections of Bor (1.3 mg/m2) on days 1, 8, 15, and 22 in combination with dexamethasone (10–20 mg) on days 1 and 2 after Bor infusion, every 5 weeks. After BD induction, Bor maintenance was initiated with a schedule of biweekly or longer intervals. The dose of BD could be reduced based on AEs or other social aspects at the discretion of the treating physician. Overall survival (OS) was defined as the period between BD initiation of treatment to the last follow-up. Results: The median age of patients at the time of BD induction was 72.6 years (range, 49–84 years), and the subtypes of MM were the IgG type in 14 patients, IgA type in 6 patients, and BJP in 2 patients. Twelve patients were men and 10 were women. On the basis of the International Staging System classification, the clinical stage was I in 3 patients, II in 16 patients, and III in 3 patients. The median number of prior treatment regimens was 2.0 (range, 1–6). The median follow-up duration was 4.3 years (inter-quartile range, 3.0–7.8 years). Ten patients were alive, and 12 patients had died at the time of reporting. The median duration from the start of the first line treatment to BD initiation was 1.8 years (range, 0.1–14.8 years). The median number of BD induction cycles was 2.0 (range, 1–6), and 8 patients (36%) achieved a partial response (PR), although the other 14 patients (64%) showed stable disease or only a minimal response. Although AEs such as PN and gastrointestinal tract symptoms developed in 10 of 22 patients during BD induction, all 22 patients could move on to the maintenance phase. The median duration of Bor maintenance was 14.5 months (range, 1.8–76.5 months). Three patients were still receiving maintenance therapy at the time of reporting. Six patients (27.2%) received maintenance therapy for over 2 years, and 3 (13.6%) received it for over 4 years. The median OS was 3.6 years (range, 0.4–6.5 years), and the duration of Bor therapy from induction to the last administration was 2.5 years. The median progression free survival was 1.6 years (range, 0.3–6.5 years). During maintenance therapy, 5 patients achieved a PR. The reasons for cessation of Bor maintenance therapy were AEs in 8 patients and progressive disease in 11 patients. No patient died during Bor administration in this study. Discussion: We retrospectively analyzed 22 TI-RR-MM patients receiving modified BD therapy. Pantani L et al. reported the results of using BD therapy for 85 RR-MM patients, and demonstrated that twice-weekly Bor administration resulted in a clinical response rate of 19% and a median OS of 22 months (Ann Hematol, 2014). Although the clinical response rate in our study was inferior to that of Pantani L et al., the median OS in our study was superior (3.6 years) than those of previous reports. Because the majority of patients in this study were frail or elderly, the early decision to change to maintenance therapy before they developed severe AEs resulted in a good clinical outcome and a longer OS. Continuing BD therapy without a satisfactory molecular response might be an attractive therapeutic approach for TI-RR-MM patients. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a neoplasm of plasma cells that is often fatal, despite the use of high dose chemotherapy and hematopoietic stem cell transplantation. Although new therapeutic approaches, including novel agents such as thalidomide, lenalidomide, and bortezomib (a proteasome inhibitor), are now used clinically and have improved the outcome of patients with MM, most patients eventually relapse, and it remains an incurable disease. Wnt/β-catenin signaling plays a critical role in both cell proliferation and differentiation in normal tissue. β-catenin, a key player and downstream effector in canonical Wnt signaling, is involved in the regulation of cell fate, proliferation, and self renewal of stem cells. The activation of Wnt signaling is aberrantduring the pathogenesis of various malignant neoplasms, and it has been suggested that Wnt/β-catenin signaling is involved in the regulation of cancer stem cells. When the Wnt signaling pathway is activated, stabilized β-catenin translocates to the nucleus, where it interacts with T-cell factor, followed by transcription of target genes such as c-myc, cyclin D1 and survivin. Nuclear β-catenin also recruits the co-activator, cyclic AMP response element-binding protein (CBP), which is essential for hematopoietic cell proliferation and hematopoietic stem cell self renewal, or its related homolog p300, which is involved in hematopoietic cell differentiation. Recent studies have shown that CBP and p300 have distinct functions in the regulation of β-catenin expression: CBP promotes β-catenin expression, whereas p300 inhibits β-catenin expression. In the context of hematological malignancies, β-catenin is overexpressed in myeloma-derived cell lines and primary myeloma cells, whereas expression is very low in normal plasma cells. Myeloma cells are maintained by several growth factors and cytokines, including Wnt ligands secreted by stromal cells in the bone marrow. These data suggest that Wnt/β-catenin signaling contributes to the pathogenesis of MM and thus might be a promising target for the treatment of this incurable hematological malignancy. Moreover, it has been reported that CBP, rather than p300, plays an important role in the expression of apurinic endonuclease/redox factor-1, an important regulator of multidrug resistance, in retinoic acid-induced chemo-resistant myeloma cells, suggesting that CBP is involved in the acquisition of drug resistance. Taken together with the previous data, Wnt/β-catenin signaling, especially CBP, might be an attractive target for new therapeutic agents against MM. ICG-001, small-molecule inhibitor of the canonical Wnt signaling pathway, specifically binds to CBP, thereby disrupting CBP/β-catenin interaction. In the present study, we investigated the role of Wnt/β-catenin signaling in myeloma cells using ICG-001. MTS and trypan blue dye exclusion assays showed that ICG-001 inhibits the proliferation of U266, RPMI8226, and KMS myeloma cell lines in a dose- (0-15 μM) and time- (0-72 h) dependent manner. Assays for apoptotic cell death were performed to determine the cause of growth inhibition by ICG-001 and demonstrated that ICG-001 induced both early and late apoptosis in myeloma cells. To investigate the molecular mechanisms of ICG-001-induced cell death in myeloma cells, the expression of various cell-death associated proteins and down-stream molecules of Wnt/β-catenin signaling was examined. Western blotting analysis showed that ICG-001 arrested cell growth and induced apoptotic cell death in myeloma cells by reducing the expression of three β-catenin target molecules: survivin, cyclin D1, and c-Myc. We next examined the effects of ICG-001 on bortezomib (BTZ)-resistant MM cells. BTZ resistance is an urgent issue in clinics, and therapeutic approaches for overcoming BTZ resistance are important. Interestingly, ICG-001 inhibited the proliferation of both BTZ-sensitive wild-type KMS and BTZ-resistant KMS cells, suggesting that targeting CBP may overcome BTZ-resistance. Furthermore, when combined with cyclosporine, which inhibits non-canonical Wnt/β-catenin signaling, ICG-001 synergistically induced the growth arrest of myeloma cells by inducing apoptotic cell death. These results indicate that inhibition of Wnt/β-catenin signaling may be an attractive therapeutic option both for patients with newly diagnosed MM and for those in a refractory or relapsed state. Disclosures No relevant conflicts of interest to declare.

Although the introduction of bortezomib and immunomodulatory drugs (IMiDs) has led to improved outcomes in patients with multiple myeloma (MM), the disease remains incurable. Bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has resulted in marked therapeutic effects; however, this therapy is often complicated by peripheral neuropathy (PN), of which grade ≥3 PN is dose-limiting toxicity and can necessitate cessation of therapy. Subcutaneous administration of bortezomib can reduce the incidence of PN; however, among cases of PN that still occur, 24% are grade 2 PN and 6% are grade 3 PN. These data suggest that the incidence of PN higher than grade 2 is not attenuated by the subcutaneous delivery of bortezomib. In addition, patients often become refractory to bortezomib after long-term use. In an effort to identify potent and well-tolerated agents, clinical trials of novel agents (e.g., carfilzomib, pomalidomide, and monoclonal antibody against CS-1) are being conducted both in patients with newly diagnosed MM and in those with relapsed/refractory disease. We previously reported that 1’-acetoxychavicol acetate (ACA) obtained from the rhizomes of the plant Languas galanga induces cell death of MM cells in vitro and in vivo through inhibition of NF-κB-related functions (Cancer Res, 2005; 65: 4417). Subsequently, we developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis to develop more potent NF-κB inhibitors, and successfully synthesized a novel benzhydrol-type analog of ACA, named TM-233, that exerted potent growth inhibition against various MM cells (U266, RPMI8226, and MM-1S cells) in a dose- and time-dependent manner when compared with ACA (Chem Pharm Bull., 2008; 56: 1490). Further, TM-233 inhibited constitutive phosphorylation of JAK2 and STAT3 and down-regulated the expression of anti-apoptotic Mcl-1 protein. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter. Mcl-1 is the downstream molecule of STAT3; therefore, these results suggest that TM-233 induces cell death in MM cells with down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, we examined the DNA-binding activity of NF-κB in TM-233-treated MM cells and found that NF-κB was inhibited by TM-233. Further, Western blotting showed that TM-233 rapidly decreased the nuclear expression of NF-κB but increased the accumulation of NF-κB in the cytosol, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. Immunohistochemical analysis confirmed that the p50/RelA dimer of NF-κB was located in the cytosol and not in the nucleus in TM-233-treated MM cells. We then examined the effects of TM-233 on bortezomib-resistant MM cells. Bortezomib-resistant MM cell lines (i.e., KMS-11/BTZ and OPM-2/BTZ) were established by limiting dilution. We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (Leukemia 2010; 24: 1506). TM-233, but not bortezomib, inhibited cellular growth and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- (0-48 hours) and dose- (0-5 μM) dependent manner. Furthermore, the combination of low-dose TM-233 (less than 2 μM) and bortezomib (10 nM) significantly induced cell death in bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting via different mechanisms from those of bortezomib. In conclusion, TM-233 induced cell death in MM cells, and this effect was mediated through the JAK/STAT and NF-κB dual-signaling pathways. These data indicate that TM-233 might be a more potent and more specific NF-κB inhibitor than that of original compound (ACA), and might be able to overcome bortezomib-resistance in MM cells. Therefore, further studies investigating clinical approaches, including combination therapy, are warranted. Disclosures: No relevant conflicts of interest to declare.

A 45-year-old woman with acute lymphoblastic leukemia (ALL) who failed to achieve complete remission (CR) after one course of induction chemotherapy with vincristine, daunorubicin, prednisolone and l-asparaginase was successfully treated with a high dose of cytosine arabinoside (Ara-C) and mitoxantrone. The leukemic blasts were CD7, 19, 33, and 38 antigens positive, and had a rearrangement in the T-cell receptor delta chain gene. The karyotype was normal. Primary induction failure and positivity for myeloid antigens are both reported to be poor prognostic factors for ALL. Nevertheless, this patient was successfully treated with the high dose Ara-C and mitoxantrone, and she remains in CR for over 20 months. Combination chemotherapy with high dose Ara-C and mitoxantrone may be of benefit for refractory ALL with both CD7 and myeloid antigens.

Abstract 2939 Multiple myeloma (MM) remains incurable despite the use of conventional and high-dose chemotherapy with stem cell transplantation. However, novel agents such as thalidomide, lenalidomide, and bortezomib have already achieved promising clinical results. In particular, bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has remarkable clinical effects in both refractory/relapsed and newly diagnosed patients. However, acquired drug resistance and serious toxicities such as peripheral neuropathy are often triggered by bortezomib in the clinical settings. Therefore, novel targeted agents with less toxicity are necessary to overcome bortezomib-resistance and improve clinical outcomes in MM. We have previously reported that 1'-acetoxychavicol acetate (ACA), a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga, has an inhibitory effect on NF-κB, and induces cell death in MM cells both in vitro and in vivo (Cancer Res, 2005; 65: 4417). With the aim of discovering more potent NF-κB inhibitors, we subsequently developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis. This showed that a novel benzhydrol-type analog of ACA (TM-233) exhibited greater inhibition of NF-κB activity in MM cells when compared with ACA. In the present study, we examined the effects of TM-233 on various MM cells, including those resistant to bortezomib and we investigated the molecular mechanism of TM-233-induced cell death in these cells. TM-233 inhibited cell proliferation and induced cell death in various MM cell lines (U266, RPMI8226, and MM-1S cells) in a time (0–48 hours)- and dose (0–5 μM)- dependent manner. Similarly potent activity was observed against primary MM cells. Exposure of bone marrow mononuclear cells from 3 patients with MM to TM-233 revealed selective activity under the same conditions as for cell lines. Moreover, TM-233 did not inhibit colony formation in CD34-positive cells from human umbilical cord blood; therefore, TM-233 did not influence the normal blood cells. We next investigated the effects of TM-233 on the various signaling pathways including JAK/STAT and NF-κB in MM cells. TM-233 activated apoptosis-related caspase-3, -8, -9, and PARP, suggesting that cell death related to TM-233 is mediated by both mitochondrial- and Fas-dependent pathways. Furthermore, treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not that of Bcl-2 and Bcl-xL proteins. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter, suggesting that TM-233-induced cell death in MM cells down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, the DNA-binding activity of NF-κB in MM cells was inhibited by the treatment of TM-233. Further, TM-233 rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. These results indicate that TM-233 induced cell death in MM cells occurs via both JAK/STAT and NF-κB pathways. We finally examined the effects of TM-233 on bortezomib-resistant MM cells. We have recently established the bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ (Leukemia 2010; 24: 1506). We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome b5 subunit, resulting in bortezomib-resistance mediated through prevention of the accumulation of unfolded proteins and fatal ER stress. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- and dose-dependent manner. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting according to a different mechanism from that of bortezomib, possibly inhibition of the JAK/STAT pathway. In conclusion, TM-233, a novel analog of ACA, might be a more potent NF-κB inhibitor than ACA and could overcome bortezomib-resistance in MM cells. Further studies investigating clinical applications are necessary and may yield interesting results. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria. Kizaki:Janssen Pharmaceutical K.K.: Honoraria.

Treatment of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid (ATRA) was associated with rapid improvement in hemostatic markers. We made serial analyses of various hemostatic parameters in seven newly diagnosed APL patients. In all patients at diagnosis, plasma fibrinogen/fibrin degradation product (fragment-E), cross-linked fibrin degradation product (D-dimer fragment), thrombin-antithrombin III complex and plasmin-α2-plasmin inhibitor complex were elevated, indicating the presence of disseminated intravascular coagulation (DIC). Antithrombin III (A TIII) levels were normal in all patients except for the patient with congenital ATIII deficiency. In four patients subsequently treated with ATRA without anticoagulant therapy, these hemostatic markers returned to near-normal levels by day 7 of treatment, indicating that DIC was essentially resolved. By contrast, in three patients who received conventional chemotherapy with a continuous low-dose heparin, improvement of coagulopathy was slower than in patients treated with ATRA. These results suggest that ATRA therapy exerts the rapid improvement in abnormal hemostatic markers in APL patients without any anticoagulant therapies, by inducing differentiation of leukemic cells and, in turns no massive release of procoagulant or fibrinolytic substances from these cells. © 1994 Wiley-Liss, Inc.

Abstract 3837 Poster Board III-773 Multiple myeloma (MM) is a plasma cell malignancy that remains incurable despite the use of conventional chemotherapy. Recent advances in the field of cell biology could lead to new therapeutic agents based upon molecular targeting, such as thalidomide and proteasome inhibitor bortezomib. These agents have remarkable activity against both newly diagnosed and refractory patients with MM, but prolonged exposure to them may result in the development of de novo drug resistance. Thus, it is necessary to identify and validate novel agents with less toxicity to overcome drug resistance and to improve the clinical outcome of MM. Progression and chemoresistance are thought to involve IL-6, whose expression is induced by NF-κB, through its regulation of the growth and survival of MM cells. IL-6 leads to constitutive activation of STAT3, which in turn results in the expression of high levels of Mcl-1. Thus, the constitutive activation of both NF-κB and STAT3 plays an important role in MM cell proliferation, and both molecules are important targets for the treatment of MM. Auranofin (AF: RIDAURA®; GSK) is a coordinated gold compound that has been widely used for the treatment of rheumatoid arthritis based on its anti-inflammatory properties through the inhibition of NF-κB activation. Therefore, assuming that AF has the potency to induce apoptosis in MM cells by interfering with NF-κB and STAT pathways, it may become a candidate for a novel therapeutic agent. To address our hypothesis, the effects of AF on inducing apoptosis of various MM cells were examined. Further, the molecular mechanism of AF-induced apoptosis in MM cells was investigated. AF inhibited the growth of U266 cells in a time- and dose-dependent manner with IC50 of 50 nM at 24 h. AF significantly induced cell cycle arrest at the G1 phase and subsequent apoptosis of U266 cells. AF-induced apoptosis in various human MM cell lines and CD138-positive plasma cells from patients with MM involved the activation of caspases-3, -8, and -9. Treatment with AF inhibited the constitutive and IL-6-induced activation of STAT3, and then downregulated the expression of Mcl-1 but not that of Bcl-2 or Bcl-xL proteins. To clarify the biological significance of Mcl-1 in AF-induced apoptosis of MM cells, Mcl-1 expression vector (pEGFP-hmcl-1) and control vector were introduced into U266 cells (designated as U266/mcl and U266/neo cells). Induction of apoptosis by AF was abrogated in U266/mcl, but not in U266/neo cells. We next examined the effect of AF on the DNA binding activity of STAT3. Electrophoretic mobility gel shift assay (EMSA) using U266 nuclear extracts demonstrated that IL-6-induced STAT3 binding activity was inhibited by the presence of AF. These results suggest that AF inhibits the IL-6-induced JAK/STAT pathway selectively and induces apoptosis in MM cells via the downregulation of Mcl-1. In addition, AF downregulated the activation of NF-κB in an IκB-independent manner, and also inhibited DNA binding activity in U266 cells. Although NF-κB inhibitory peptide (SN-50) did not directly induce the expression of Mcl-1, the combination of SN-50 with AF reduced the levels of Mcl-1, suggesting that inhibition of NF-κB potentiates the apoptotic effect of AF. Finally, our in vitro data prompted us to examine whether or not the effects of AF are equally valid in a clinical settings. The protocol for assessing the clinical benefit of oral administration of AF (RIDAURA®) in patients with relapsed or refractory MM was approved by the Ethics Committee of Saitama Medical University. Since 2008, 7 patients with refractory MM were treated with oral RIDAURA® 6 mg daily. The median follow-up for patients was 7.5 months. All 7 patients responded to AF, including one PR (partial response) and 6 SD (stable disease) with no progression. No adverse events have been observed to date. Details of the clinical outcome will be presented. In conclusion, gold compound AF inhibited constitutive activation of both STAT3 and NF-κB, resulting in the downregulation of anti-apoptotic Mcl-1 protein in MM cells with clinical relevance. A low pharmacological concentration (50 nM) of AF is widely employed for the treatment of rheumatoid arthritis without any side effects; therefore, it may be used to treat MM without the risk of severe toxicity. We propose that AF (RIDAURA®) may have potential use as a new molecular-targeted agent for the treatment of MM. Disclosures: Off Label Use: Auranofin in the treatment of multiple myeloma.

Resistance to activated protein C (APC), recently reported to be the most prevalent inherited cause of thrombosis among Caucasians, is associated with a single point mutation in the coagulation factor V gene. We investigated the prevalence of APC resistance and the factor V gene mutation (R506Q) in 34 consecutive Japanese patients with venous thrombosis or pulmonary thromboembolism and 63 control subjects. Three of the 33 patients examined (9%) had an APC ratio below the 5th percentile of control values (2.27), but all were above 2.0. The factor V mutation (R506Q) was not detected in the 29 patients studied, including the 3 patients whose APC ratios were below 2.27, or in 53 controls. In a tissue factor-based factor V assay to detect APC resistance recently described by Le et al. (Blood 1995;85:1704-1711), all patients studied were found to be normal including the three with a low APC ratio. We conclude that APC resistance and factor V gene mutation are less prevalent in Japan than in several European countries.