Durable Remission of Chemotherapy-Refractory Myeloid Sarcoma by Azacitidine.

Myeloid sarcoma is a rare disease entity of extramedullary myeloid neoplasm that can occur both as an initial isolated myeloid sarcoma without leukemic cell invasion in the peripheral blood and bone marrow, and as the secondary lesion of acute and chronic myeloid leukemias, myelodysplastic syndrome and chronic myeloproliferative neoplasms. Due to its rarity and its frequent emergence as the recurrent lesion after intensive systemic therapy, including allogeneic hematopoietic stem cell transplantation, the standard treatment has not been established for myeloid sarcoma. In this report, we presented an 84-year-old female patient with isolated myeloid sarcoma which progressed to myelodysplastic syndrome and systemic myeloid sarcoma despite various types of conventional anti-leukemic chemotherapies. However, the patient got a durable partial response by the monotherapy of azacitidine, a hypomethylating agent. She received thirteen courses of azacitidine therapy without progression. We discuss the possibility that hypomethylating agents are the novel effective and feasible therapeutic options for myeloid sarcoma, even in cases refractory to or relapsed after intensive systemic treatment. We also discuss the possible future development of hypomethylating agent-containing combinatory therapeutic strategy for myeloid sarcoma, given its direct anti-leukemic effect and immunomodulatory effect.

Sequential therapy of four cycles of bortezomib, melphalan, and prednisolone followed by continuous lenalidomide and dexamethasone for transplant-ineligible newly diagnosed multiple myeloma.

The combinations of melphalan, bortezomib, and prednisolone (VMP) and of lenalidomide and dexamethasone (Rd) are standard treatment strategies for transplant-ineligible newly diagnosed multiple myeloma (NDMM). To make the most of these two strategies, we investigated the efficacy and feasibility of first-line treatment with 4 cycles of VMP followed by continuous Rd therapy in a multi-institutional phase 2 study in Japanese patients with transplant-ineligible NDMM. Thirty-six patients of median age 74 years old with NDMM initially received 35-day cycles of VMP: oral melphalan (6 mg/m2) and prednisolone (60 mg/m2) on days 1 to 4 and bortezomib (1.3 mg/m2) on days 1, 8, 15, and 22. After 4 cycles of VMP, treatment was switched to 28-day cycles of Rd, which was continued until disease progression or emergence of an unacceptable adverse event (AE) in 33 patients, while one patient who achieved CR after VMP continued VMP at the physician's discretion. The overall response rates after VMP and after Rd were 66.7% and 86.1%, including CR rates of 5.6% and 36.1%, respectively. In a median follow-up period of 34.3 months, the progression-free survival and overall survival rates at 3 years were 43.2% and 81.3%, respectively. Grade 3-4 hematological AEs included neutropenia (39% with VMP and 24% with Rd) and thrombocytopenia (11% with VMP and 3% with Rd). There was no death due to an AE. In conclusion, sequential therapy with VMP followed by Rd is effective and mostly feasible for transplant-ineligible NDMM. The study is registered as UMIN000034815.

Retrospective analysis of primary plasma cell leukemia in Kansai Myeloma Forum registry.

We retrospectively analyzed twenty-six patients with primary plasma cell leukemia (pPCL) registered from May 2005 until April 2015 by the Kansai Myeloma Forum. Twenty patients received novel agents (bortezomib or lenalidomide), and their median survival of was 34 months. The median survival of patients who underwent autologous stem cell transplantation (SCT) was 40 months, those undergoing allogeneic SCT 55 months, and those undergoing both types of SCT (auto-allo) 61 months; whereas for those who did not undergo SCT it was 28 months (p = 0.845). The only statistically significant risk factor identified by multivariate analysis was hypercalcemia.

Efficacy and safety of obinutuzumab in patients with previously untreated follicular lymphoma: a subgroup analysis of patients enrolled in Japan in the randomized phase III GALLIUM trial.

GALLIUM is a global phase III study that demonstrated a statistically significant and clinically meaningful improvement in progression-free survival (PFS) with obinutuzumab plus chemotherapy (G-chemo) versus rituximab plus chemotherapy (R-chemo) in previously untreated patients with follicular lymphoma (FL). In this single-country subgroup analysis, we explored patterns of efficacy and safety in patients enrolled in the GALLIUM study in Japan (Japanese subgroup). Patients were randomized to open-label induction treatment with G-chemo or R-chemo. Responders received maintenance monotherapy with their randomized antibody for up to 2 years. The primary endpoint was investigator-assessed PFS. Overall, 123 patients with FL were randomized in the Japanese subgroup (G-chemo, n = 65; R-chemo, n = 58). The majority of patients received cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (82.9 vs 33.1% in the global GALLIUM FL population). PFS at 3 years was 89.9% (G-chemo) vs. 74.7% (R-chemo); hazard ratio 0.42; 95% confidence interval 0.15, 1.15; P = 0.08. Higher rates of grade 3-5 adverse events (96.9 vs. 89.7%) and serious adverse events (35.4 vs. 22.4%) were observed with G-chemo vs R-chemo, respectively. Neutropenia was frequent in the Japanese subgroup (92.3% G-chemo; 79.3% R-chemo). Overall, the results in the Japanese subgroup were consistent with those in the global GALLIUM population.

Pomalidomide with or without dexamethasone for relapsed/refractory multiple myeloma in Japan: a retrospective analysis by the Kansai Myeloma Forum.

Determinants of the efficacy and safety of pomalidomide (POM) monotherapy or POM plus dexamethasone (DEX) (POM/DEX) for relapsed and refractory multiple myeloma (RRMM) were examined retrospectively in a real-world clinical practice setting in Japan. The subjects were 108 patients registered with the Kansai Myeloma Forum, who were treated with either POM or POM/DEX. Of these, 79 (73%), 73 (68%), and 58 (54%) were resistant to bortezomib (BTZ), lenalidomide (LEN), and both BTZ and LEN, respectively. The median overall survival (OS) was not reached. The median time to treatment failure (TTF) was 4.4 months. The best response was recorded in 96 patients, with a 31% overall response rate (ORR) and a 79% rate of achieving at least stable disease. Number of pre-POM regimens ≥ 5, non-IgG-type M-protein, and time from initial therapy to POM or POM/DEX therapy < 2 years were associated with shorter TTF and OS. Frequent (> 10%) severe adverse events included neutropenia (55.1%), thrombocytopenia (33.7%), anemia (30.6%), febrile neutropenia (12.2%), fatigue (11.2%), and anorexia (10.2%). In conclusion, POM and POM/DEX showed substantial efficacy against RRMM, but new combination therapies with POM are needed to improve efficacy further without causing hematologic toxicities.

Second primary malignancies among patients with myeloma-related-diseases in the KMF database.

The incidence of second primary malignancies (SPMs) in Japanese patients with myeloma or myeloma-related diseases was studied by using the Kansai Myeloma Forum (KMF) database registered from November 2012 to March 2015. We studied 1,571 cases. Hematologic malignancies were documented in 10 patients, and solid tumors in 36 during this period. The cumulative 5-year incidence was estimated to be 1.0% for hematological malignancies and 3.7% for solid tumors. In the patients with smoldering myeloma or MGUS without treatment, solid tumors but not hematologic malignancies developed, though the cumulative incidence of each malignancy did not differ significantly from that in patients receiving treatment. Although statistical analysis showed that treatment with melphalan, bortezomib, lenalidomide, or thalidomide had no effect on the occurrence of hematological malignancies, lenalidomide administration was more frequent in the patients with solid tumors. To evaluate the SPMs in myeloma or myeloma-related diseases more accurately, accumulation of a larger number of patients and longer observation are needed.

The response to second-line induction with bortezomib and dexamethasone is predictive of long-term outcomes prior to high-dose chemotherapy with autologous stem cell transplantation for multiple myeloma.

OBJECTIVE: We retrospectively investigated the efficacy and predictive factors for the treatment outcomes of bortezomib plus dexamethasone (BD) as second-line induction therapy prior to high-dose chemotherapy supported by autologous stem cell transplantation (HDT/ASCT) in multiple myeloma (MM) patients. METHODS: Sixty-six transplant eligible MM patients treated by the Kyoto Clinical Hematology Study Group between 2006 and 2011 were investigated. Conventional induction chemotherapy, including vincristine, doxorubicin and dexamethasone (VAD) and high-dose dexamethasone (HDD), was used as first-line induction therapy in all patients, seven (10.6%) of whom attained a very good partial response (VGPR). Of the 59 patients who did not attain VGPR with VAD or HDD, 33 were given BD as second-line induction therapy prior to HDT/ASCT. RESULTS: Patients not treated with BD induction showed an overall response rate (ORR, i.e., better than partial response) of 85.3% after induction therapy, while the ORR of patients treated with BD induction improved from 42.4% after conventional induction therapy to 84.8% after BD. The overall survival (OS) and progression-free survival (PFS) of patients not treated with BD induction were not significantly influenced by the response to induction therapy. Among the patients treated with BD, failure in attaining VGPR prior to ASCT was associated with a significantly shorter PFS and it also tended to show a shorter OS, while the disease stage and achievement of a complete response after HDT/ASCT had no impact on OS or PFS. CONCLUSION: The achievement of at least VGPR with second-line BD induction therapy is a prerequisite for attaining longer OS and PFS after HDT/ASCT.

Bortezomib plus dexamethasone for relapsed or treatment refractory multiple myeloma: the collaborative study at six institutes in Kyoto and Osaka.

We conducted a retrospective collaborative investigation of bortezomib (Bor) plus dexamethasone (Dex) therapy (BD Tx) for 88 relapsed or refractory (Rel/Ref) MM patients at six institutes. One cycle BD Tx comprised of Bor (1.3 mg/m²/day) on days 1, 4, 8 and 11, and Dex on days 1, 2, 4, 5, 8, 9, 11 and 12, every 21 days, and the mean number of BD Tx cycles was 3. The overall response rate was 66.9%, the median overall survival (OS) was 510 days, and the median progression-free survival (PFS) was 113 days. Attainment of partial response (PR) with the first course of BD Tx associated with the longer OS and PFS and late good responder, while no patient who did not achieve PR with the first cycle attained better than very good PR (VGPR) with the subsequent BD Tx. Patient age of less than 64 years old also associated with the longer OS and PFS. In addition, both an earlier disease stage and Dex dosage had a significant impact on OS, while the attainment of VGPR within 2 cycles had a significantly longer PFS. Earlier BD Tx courses may be predictive for the subsequent therapeutic pathway of Rel/Ref MM.

Prognostic significance of the BAALC isoform pattern and CEBPA mutations in pediatric acute myeloid leukemia with normal karyotype: a study by the Japanese Childhood AML Cooperative Study Group.

High BAALC (brain and acute leukemia, cytoplasmic) gene expression may indicate an adverse prognosis for adults who have acute myeloid leukemia (AML) and a normal karyotype, but its prognostic significance for pediatric AML cases is unclear. Whether different BAALC isoform patterns are of prognostic significance is also unclear. Newly diagnosed AML patients with normal karyotype who were treated by the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed in terms of their BAALC expression levels (n = 29), BAALC isoforms (n = 29), and CEBPA mutations (n = 49). Eleven and 18 patients exhibited high and low BAALC expression, respectively, but these groups did not differ significantly in terms of overall survival (54.6 vs. 61.1%, P = 0.55) or event-free survival (61.4 vs. 50.0%, P = 0.82). Three of these 29 patients (10.3%) expressed the exon 1-5-6-8 BAALC isoform along with the expected 1-6-8 isoform and had adverse clinical outcomes. Novel CEBPA mutations were also identified in four of 49 patients (8.2%). All four patients have maintained complete remission for at least 5 years. Thus, 1-5-6-8 isoform expression may be associated with an adverse prognosis in pediatric AML with normal karyotype. CEBPA mutations may indicate a favorable prognosis.

Osteoclasts are involved in the maintenance of dormant leukemic cells.

Osteoclasts (OCs) are specialized cells for the resorption of bone matrix that have also been recently reported to be involved in the mobilization of hematopoietic progenitor cells. When Ba/F3 cells expressing wild-type bcr-abl were co-cultured with osteoblasts (OBs), OCs, and bone slices, their proliferation was significantly suppressed, and the Ki-67 negative population, which is believed to be in G(0) phase, was increased. The results of our in vitro experiments suggest that OCs could be involved in the maintenance of dormant leukemic cells in the bone marrow (BM) microenvironment through the release of soluble factors, one of which could be TGF-beta.

beta-catenin small interfering RNA successfully suppressed progression of multiple myeloma in a mouse model.

PURPOSE: beta-catenin is the downstream effector of the Wnt signaling pathway, and it regulates cell proliferation. beta-catenin overexpression correlates positively with prognosis in several types of malignancies. We herein assessed its effects on growth of multiple myeloma cells using a xenograft model. EXPERIMENTAL DESIGN: We first investigated the expression of beta-catenin in multiple myeloma cell lines and multiple myeloma cells obtained from patients. Next, we investigated the growth inhibitory effects of beta-catenin small interfering RNA on the growth of multiple myeloma cells in vivo. Six-week-old male BALB/c nu/nu mice were inoculated s.c. in the right flank with 5 x 10(6) RPMI8226 cells, followed by s.c. injections of beta-catenin small interfering RNA, scramble small interfering RNA, or PBS/atelocollagen complex twice a week for a total of eight injections. RESULTS: Significantly higher levels of beta-catenin expression were observed in multiple myeloma cell lines and in samples from patients with multiple myeloma than those found in mononuclear cells obtained from healthy volunteers. In in vivo experiments, no inhibitory effects were observed following treatment with scramble small interfering RNA or PBS/atelocollagen complexes, whereas treatment with beta-catenin small interfering RNA/atelocollagen complex significantly inhibited growth of multiple myeloma tumors (P < 0.05). CONCLUSIONS: beta-catenin small interfering RNA treatment inhibited the growth of multiple myeloma tumors in a xenograft model. To our knowledge, this is the first report showing that the treatment with beta-catenin small interfering RNA produces an inhibitory effects on growth of hematologic malignancies in vivo. Because treatment with beta-catenin small interfering RNA inhibited growth of multiple myeloma cells, beta-catenin is the attractive novel target for treating multiple myeloma.

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury.

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury.

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation.

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD-fmk did not completely rescue FK228-induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule-associated protein light chain 3 (LC3-I) to conjugated light chain 3 (LC3-II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase-independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228-induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228-induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells.

Involvement of extracellular signal-regulated kinase activation in human osteosarcoma cell resistance to the histone deacetylase inhibitor FK228 [(1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone].

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer.

Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer.

Anti-myeloma effect of homoharringtonine with concomitant targeting of the myeloma-promoting molecules, Mcl-1, XIAP, and beta-catenin.

Since a variety of cell intrinsic and extrinsic molecular abnormalities cooperatively promote tumor formation in multiple myeloma (MM), therapeutic approaches that concomitantly target more than one molecule are increasingly attractive. We herein demonstrate the anti-myeloma effect of a cephalotaxus alkaloid, homoharringtonine (HHT), an inhibitor of protein synthesis, through the induction of apoptosis. HHT significantly reduced Mcl-1, a crucial protein involved in myeloma cell survival, in all three myeloma cell lines examined, whereas certain BH3-only proteins, such as Bim, Bik, and Puma, remained unchanged following HHT treatment, and their expression levels depended on the cell type. HHT also reduced the levels of c-FLIP(L/S), activated caspase-8, and induced active truncated-Bid. Thus, HHT-induced apoptosis appears to be mediated via both intrinsic and extrinsic apoptosis pathways, and the resultant imbalance between BH3-only proteins and Mcl-1 may be pivotal for apoptosis by HHT. In addition, HHT treatment resulted in reduced levels of beta-catenin and XIAP proteins, which also contribute to disease progression and resistance to chemotherapy in MM. In combination, HHT enhanced the effects of melphalan, bortezomib, and ABT-737. These results suggest that HHT could constitute an attractive option for MM treatment though its ability to simultaneously target multiple tumor-promoting molecules.