ALBERT

Description: Background: Analysis of prognostic factors and clinical trials of novel agents for Waldenstrӧm macroglobulinemia (WM) are ongoing in Western countries, but few studies of WM have been performed in Japan. As a step toward future investigations, we retrospectively analyzed clinical features and prognostic factors in Japanese patients with WM. Methods: We retrospectively analyzed clinical and laboratory characteristics, treatment and outcomes of 110 patients with WM, IgM-MGUS or lymphoplasmacytic lymphoma (LPL) diagnosed from January 2001 to March 2013 at 12 institutes. Overall survival (OS) was analyzed using Kaplan-Meier methods and survival was compared using log-rank testing. Several clinical characteristics at diagnosis were assessed by Cox regression for uni- and multivariate analysis for OS. Results: Median age at diagnosis was 69 (range, 41-96) years, 73.6% were male, 12.0% had an ECOG performance status 2-4 and 6.4% presented with B-symptoms. Hyperviscosity, peripheral neuropathy, amyloidosis, cryoglobulinemia and cold agglutinin disease were shown in 9.1%, 4.5%, 1.8%, 4.5% and 2.7%, respectively. In 94 patients with available CT findings at diagnosis, lymphadenopathy, hepatosplenomegaly, pleural effusion, lung involvement, bone involvement and skin involvement were shown in 41.5%, 14.9%, 8.5%, 4.3%, 4.3% and 6.4%, respectively. Median serum monoclonal protein level was 2.62 g/dl (range, 0.70-9.35 g/dl). Symptomatic WM was present in 76 patients, asymptomatic WM in 23 and IgM-MGUS in 2 according to criteria of the Second International Workshop on WM. Seven patients showed IgG- or IgA-secreting LPL and 2 showed LPL without bone marrow infiltration. In patients with symptomatic WM, international prognostic scoring system for WM (ISSWM) was low in 9.2%, intermediate in 34.2%, high in 39.5% and unknown in 17.1%. Among patients with asymptomatic and symptomatic WM, watchful waiting was performed in 91.3% and 40.0%, respectively, with 61.9% and 36.7% remaining untreated, respectively. Median time to treatment from diagnosis of asymptomatic or symptomatic WM was 240 days (range, 3-1238 days) and 31 days (range, 0-2011 days), respectively. Oral alkylating agents were administered to 34.7% of patients with WM, 19.4% were treated with CHOP or CHOP-like regimen with or without rituximab, 8.2% received fludarabine mono- or combination therapy and 6.1% received rituximab monotherapy. Rituximab-containing therapy was administered as the initial treatment in 33.8% of patients who received treatment. Overall response rate (ORR) (complete + partial response rate) was 48.6%, and patients treated with rituximab-containing therapy displayed higher ORR (64.0%) compared to those with non-rituximab therapy (40.8%). Plasmapheresis was performed in 3.7% of patients. Three patients (2.7%) showed transformation to diffuse large B-cell lymphoma, and 7 (6.4%) developed second primary malignancies. Median follow-up was 38 months, 5-year OS rate for all patients was 74.9% (95% confidence interval (CI) 62.5-83.7) and rates for those with symptomatic WM, asymptomatic WM and other LPL were 66.0% (95%CI 50.6-77.6), 100% and 88.9% (95%CI 43.3-98.4), respectively. Significant differences in survival between risk groups of ISSWM in patients with symptomatic WM were not seen (5-year OS: high, 62.4%; intermediate, 64.3%; low, 75.0%; p=0.86). Although no significant difference in OS was observed compared to initial treatment (p=0.265), patients treated with rituximab during the observation period showed significantly prolonged OS compared to those treated without rituximab (5-year OS rates: 78.9% vs. 45.6%, p=0.036). In univariate analysis, age, pleural effusion, serum albumin, C-reactive protein and serum IgM levels were poor prognostic factors for OS. In multivariate analysis, age 〉65 years (hazard ratio (HR)=3.294; 95%CI 1.097-9.888, p=0.0336) and pleural effusion (HR=4.55; 95%CI 1.602-12.930, p=0.0045) were identified as significant prognostic factors for OS. Conclusion: Prognostic factors for WM in Western countries may not be applicable to Japanese patients. This study suggested presence of pleural effusion at diagnosis is associated with poor clinical outcomes. Further investigations including histopathological examinations and molecular analyses are required to elucidate prognostic factors in Japan. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: B7-H1 (also known as PD-L1 or CD274), a co-inhibitory molecule of the B7 family, is detected on various tumor cells and associated with tumor evasion from cytotoxic T lymphocyte (CTL)-mediated immune surveillance. Our previous study showed that B7-H1 expression levels on plasma cells from multiple myeloma (MM) patients were significantly upregulated compared with those cells from monoclonal gammopathy of undetermined significance patients and healthy volunteers. B7-H1-expressing MM cells had a proliferative advantage and were resistant to antimyeloma agents (Tamura et al. Leukemia 2013). However, it remains unknown whether cellular responses in B7-H1-expressing MM cells are affected by the interaction of B7-H1 molecules with their receptors, i.e., PD-1 and CD80. We thus investigated the reverse signal derived from B7-H1 binding to their receptors on MM cells and examined the clinical characteristics of B7-H1-highly positive MM patients in a multicenter study. Methods: 1) We established B7-H1-expressing MM cell lines called MOSTI expressing high levels of CD38 and CD138 from bone marrow mononuclear cells of myeloma patients and B7-H1-positive MM cells (B7-H1.KMS28PE) stably transfected with the B7-H1 gene. 2) B7-H1 knockdown MOSTI cell lines were obtained using B7-H1-specific short-hairpin RNA expressing a lentiviral vector. 3) The proliferative potential was examined by BrdU incorporation using flow cytometry (FCM) and the MTT assay. 4) Drug-induced apoptotic cells were stained with annexin V and propidium iodide (PI) and detected in FCM. 5) Magnetic Dynabeads were coupled with PD-1-Ig or CD80-Ig fusion proteins. B7-H1-expressing MM cells were co-cultured with the beads, and the binding capacity of the beads to B7-H1+ MM cells, drug sensitivity, and cell proliferation of B7-H1+ MM cells were analyzed. 6) We classified 105 cases with newly diagnosed MM into two groups according to B7-H1 expression levels on plasma cells and compared the clinical characteristics associated with prognosis between B7-H1-highly-expressing MM patients (n=43) and other patients (n=62). Results: 1) Knockdown of B7-H1 expression in MOSTI cells significantly suppressed cell proliferation and increased melphalan-induced apoptosis. These results demonstrated that B7-H1 expression is directly associated with aggressive myeloma behavior including cell growth and drug resistance. 2) B7-H1 molecules on MOSTI and B7-H1.KMS28PE cells bound more strongly to PD-1-Fc than to CD80-Fc. The binding of PD-1-Fc to MOSTI cells was inhibited by anti-B7-H1 antibody in a concentration-dependent manner. In MOSTI cells treated with PD-1-Fc beads, apoptosis induced by both melphalan and bortezomib was markedly inhibited in comparison with the cells treated with control Ig. PD-1-Fc bead-treated B7-H1.KMS28PE cells were also resistant to melphalan-induced apoptosis. However, CD80-Fc bead-treated cells did not show drug resistance. Resistance to antimyeloma agents via the reverse signal from PD-1 to MM cells was inhibited by the PI3K/AKT inhibitor LY294002. In Western blot analysis, phospho-AKT expression was significantly upregulated in PD-1-Fc-treated MM cells. However, the cell growth of PD-1-Fc-treated MOSTI cells was the same as that of control Ig-treated cells. These data indicate that the reverse signal delivered from B7-H1 expressed on MM cells bound to PD-1 induced the drug resistance of MM cells thorough the Akt signaling pathway. 3) Patients with B7-H1 highly-expressing MM cells tended to have the poor-risk cytogenetic abnormality t(4;14) (P=0.0703). Furthermore, fibroblast growth factor receptor 3 was significantly upregulated on MM cells in those B7-H1-highly positive patients (P=0.0141). Expression levels of CD56, CD45, and CD221, which were reported to be poor prognostic markers in MM, were siginificantly higher in B7-H1-highly positive patients compared with others. Conclusion: Our study revealed a new mechanism via which the interaction between B7-H1 on MM cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces the drug resistance of MM cells through the Akt signaling pathway. Furthermore, B7-H1 expression on MM cells may be associated with t(4;14) translocation and poor prognostic MM antigens. Thus, B7-H1 may be a reasonable target for immunotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The immunophenotypic analysis of plasma cells using flow cytometry (FCM) is useful for the diagnosis for multiple myeloma (MM) and the detection of MM cells after treatment. CD138 is a well-known surface marker identifying plasma cells, although decreased CD138 expression on plasma cells is frequently observed in MM patients with relapsed or progressive disease. Thus, more stable MM antigens are needed. The SLAM family molecule CD229 has recently been reported to be specifically overexpressed on MM cells including their clonogenic precursors, suggesting that it may be a good marker to identify MM cells. Furthermore, some surface antigens, i.e., costimulatory molecule CD86 (B7-2), its receptor CD28, IL-6 receptor (CD126), insulin-like growth factor-1 (CD221), and coinhibitory molecule B7-H1 (PD-L1, CD274), were reported to be associated with poor prognosis. This study aimed to validate the potential of CD229 as a new MM cell marker and the efficacy of immunophenotypes on MM cells as prognostic markers in a multicenter study. Patients & Methods: Two-hundred thirteen patients comprising 144 newly diagnosed (18 asymptomatic and 126 symptomatic) MM patients, 25 refractory/relapsed MM patients, and 44 monoclonal gammopathy of undetermined significance (MGUS) patients were enrolled. Immunophenotyping of plasma cells in bone marrow was performed with standard 3-color FCM, in which plasma cells were gated by CD38-highly positive cells and 9 parameters, i.e., expression of MM antigens (CD138, CD229) and prognosis-related antigens previously reported (CD28, CD45, CD56, CD86, CD126, CD221, CD274) on MM cells, were analyzed. Expression levels of prognosis-related antigens were compared between patients in high-risk categories, i.e., International Scoring System (ISS) stage II/III, high-risk chromosomal abnormalities [t(4:14), t(14:16), del17p] by FISH, high-risk group stratified by the International Myeloma Working Group (IMWG) [ISS stage II/II and t(4:14) and/or del17p], high serum lactase dehydrogenase (LDH) level, or revised ISS stage III, and other patients. The revised ISS is a powerful new tool to predict outcome in MM patients treated with novel agents based on 3 factors: serum LDH level, ISS stage, and high-risk chromosomal abnormalities [Oliva S, et al. EHA2014, Abstract #S1289]. Differences between continuous variables were evaluated using the Mann-Whitney U-test. Results: 1) CD229 was expressed on almost all MM cells, even on low CD138-expressing MM cells. MM cells from ISS stage II/III patients expressed significantly lower levels of CD138 than those from ISS stage I patients (P = 0.0004). Furthermore, although CD138 expression levels in symptomatic MM patients were lower than those in patients with asymptomatic MM and MGUS and the expression was decreased in refractory/relapsed MM patients, CD229 expression levels on plasma cells were similar in MGUS and MM patients. 2) Newly diagnosed MM patients with high-risk chromosomal abnormalities or in the IMWG high-risk group had higher levels of CD86 and CD126 compared with other patients (P = 0.0056 and 0.0248 in high-risk chromosomal abnormalities, P = 0.0221 and 0.0396 in IMWG high-risk group, respectively). Furthermore, patients in revised ISS stage III had higher levels of CD126 compared with others (P = 0.0646). 3) Although the serum IL-6 level was significantly higher in ISS stage II/III patients, in patients with high LDH levels, and in revised ISS stage III patients compared with other groups, there was no correlation between serum IL-6 levels and expression levels of the IL-6 receptor CD126 on MM cells. Conclusion: CD229 has the potential to become a new marker for the identification of MM cells and target for immunotherapy. High expression levels of CD86 and CD126 were associated with high-risk patients, and CD126 may be associated with survival in patients treated with novel agents. FCM analysis may be useful for predicting prognosis as well as detecting malignant clones. Further studies are in progress to clarify the significance of those molecules on MM cells. Disclosures No relevant conflicts of interest to declare.

Description: Background: We recently reported that the International Prognostic Scoring System for Waldenström macroglobulinemia (ISSWM), which is widely used to predict the prognosis of WM patients, might not be applicable to Japanese patients, and evidence of pleural effusion might be a novel adverse prognostic factor for symptomatic WM in the rituximab era. Further studies with a large number of patients are deemed to be conducted. Methods: We retrospectively analyzed the clinical data of 498 patients with WM diagnosed between January 2001 and December 2015 from 44 institutes involved with the Japanese Society of Myeloma. The overall survival (OS) was analyzed using Kaplan-Meier methods and compared using log-rank test. Several clinical characteristics at the diagnosis were assessed by Cox regression for univariate and multivariate analyses of the OS. Results: We included 420 cases diagnosed with symptomatic (n=314) and asymptomatic WM (n=106) in accordance with the classification of the Second International Workshop on WM. The median age at the diagnosis was 69 (range, 32-91) years, with 75.5% male, and 16.0% had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 2-4. Oral alkylating agents, purine analogs, cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) or CHOP-like regimens ± rituximab, rituximab monotherapy, or dexamethasone, rituximab and cyclophosphamide (DRC) were mainly administered as initial treatment. Rituximab-containing therapy was administered in 76.8% of all patients. The median follow-up was 45 months. The 5-year OS rate for all patients was 77.9%, while the rates for those with symptomatic and asymptomatic WM were 72.9% and 92.2%, respectively. Significant differences in the survival were seen between risk groups of ISSWM in symptomatic WM patients (5-year OS: high, 55.4%; intermediate, 81.2%; low, 90.2%; p65 years, platelet count ≤10×104/µL, serum β2-microglobulin (β2-MG) 〉3 mg/L, ECOG PS 2-4, abnormal karyotype, pleural involvement, WBC 1.5 mg/dL, CRP 〉2.0 mg/dL and sIL-2R 〉4000 U/mL were significant adverse prognostic factors for the OS. A multivariate analysis revealed that a platelet count ≤10×104/µL (hazard ratio [HR] 5.942; 95% confidence interval [CI] 2.265-14.761), serum β2-MG 〉3 mg/L (HR 2.748; 95% CI 1.091-7.655), ECOG PS 2-4 (HR 2.899; 95% CI 1.219-6.290), and pleural involvement (HR 11.066; 95% CI 3.672-29.829) were adverse independent risk factors for symptomatic WM. We constructed a prognostic model by combining these prognostic variables as follows: patients with good risk (n=219), no adverse factors or only serum β2-MG 〉3 mg/L or ECOG PS 2-4; patients with poor risk (n=81), ≥1 adverse factors with a platelet count ≤10×104/µL, pleural involvement, or both serum β2-MG 〉3 mg/L and ECOG PS 2-4. The 5-year OS rates were 82.3% for good risk and 44.4% for poor risk, and this prognostic model significantly stratified symptomatic WM patients separately by the survival (p

Description: We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 × 10−9 to 4 × 10−11 mol/L when using the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 × 10−11mol/L; in contrast, the 1,25D3 produced an ED50of 10−9 mol/L with the HL-60 target cells. Ro 25-9716 (10−9 mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10−8 mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1v 48% of the untreated control cells). The p27kip-1, a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10−7 mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 × 10−11mol/L) and their expression of CD11b was enhanced (80% positive [10−9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10−9mol/L) and all-trans retinoic acid (ATRA, 10−7 mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 × 10−9 mol/L] and Kasumi-1 [ED50, 5 × 10−10 mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27kip-1. In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

Description: We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 × 10−9 to 4 × 10−11 mol/L when using the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 × 10−11mol/L; in contrast, the 1,25D3 produced an ED50of 10−9 mol/L with the HL-60 target cells. Ro 25-9716 (10−9 mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10−8 mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1v 48% of the untreated control cells). The p27kip-1, a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10−7 mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 × 10−11mol/L) and their expression of CD11b was enhanced (80% positive [10−9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10−9mol/L) and all-trans retinoic acid (ATRA, 10−7 mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 × 10−9 mol/L] and Kasumi-1 [ED50, 5 × 10−10 mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27kip-1. In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

Description: Patients diagnosed with polycythemia vera (PV) or essential thrombocythemia (ET), a subtype of myeloproliferative neoplasms (MPN), sometimes suffer disease transformation into myelofibrosis (MF) associated with poorer prognosis. Thus, predicting which patients have a risk of MF transformation is an important task. Following the identification of a driver mutation JAK2V617F in a majority of MPN patients, several studies was performed to investigate the potential of JAK2V617F allele burden as a diagnostic marker for MF-transformation. However, the results differ between cohorts presumably due to a lack of accurate JAK2V617F allele burden measurement. Since we have previously developed alternative-binding probe competitive polymerase chain reaction (ABC-PCR) that accurately determines the JAK2V617F allele burden, we assessed the predictive value of JAK2V617F allele burden in MF-transformation in Japanese MPN cohort. In a retrospective study, we compared JAK2V617F allele burdens between formalin-fixed paraffin embedded-bone marrow (FFPE-BM) from initial diagnosis and peripheral blood (PB) from follow-up visits. We first examined whether the allele burdens in FFPE-BM and PB were comparable when they are collected at the same time. Determining the allele burdens in a set of FFPE-BM and PB taken from same patient within a 3-month period, we observed that allele burdens from these specimens are significantly correlated (n=26, R²=0.97), which is consistent with previous report with a larger cohort (Blood 122; 3784-6). Thus, in subsequent analyses, we set a base line of the mutant allele burden determined from FFPE-BM, which is then compared with allele burdens from PB during the disease duration. We examined 14 PV and 20 ET patients (mean disease duration 69.2 months) defined by WHO 2008 MPN criteria. From first diagnosis to the time when MF-transformation was first recognized, JAK2V617F allele burden was significantly increased (mean increase 19.5±17.3%, p=0.044) in patients with MF-transformation (n=11). While patients with no MF-transformation (n=23) presented limited changes (mean increase 3.9±16.1%) over a similar duration period. When subclassifying patients into three groups based on the change or the base line value of JAK2V617F allele burden, MF-transformation was more frequently (p=0.034) observed in patients whose JAK2V617F allele burden was either increased by more than 10% during the follow-up (group A) or higher at first diagnosis than the mean values for each disease (PV; 71.7%, ET; 35.5%) (group B). MF-transformation was 2 out of 4 (50%) in the group A, and 9 out of 22 (41%) in the group B. In contrast, MF transformation in the rest of the patients (group C) was 0 out of 8 (0%). Hydoxyurea-treated (n=16, 6 PV and 10 ET) and –untreated (n=18, 8 PV and 10 ET) patients do not show significant difference in frequencies of MF-transformation, confirming that Hydoxyurea has no preventative effect against MF-transformation. In conclusion, our study showed that higher JAK2V617F allele burden at first diagnosis or a dynamic increase in allele burden during the follow-up period is a predictive factor for MF-transformation. Thus, a routine measurement of the JAK2V617F allele burden by an accurate assay system is recommended to predict MF-transformation. Disclosures No relevant conflicts of interest to declare.