Kooperativer Bibliotheksverbund

In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 2673-2673

Abstract: Abstract 2673 Introduction: Follicular Lymphoma International Prognostic Index 2 (FLIPI-2) is a widely accepted tool for risk assessment of follicular lymphoma (FL), which is based on age, hemoglobin level, presence of bone marrow (BM) invasion, tumor size, and b2-microgloblin levels. Although it is easy to evaluate in clinical practice, it is a combination of tumor burden and patient physical condition, and a simple and powerful biomarker reflecting the tumor burden and its character is still not established. LR11 (also called SorLA or SORL1) was identified and characterized as a regulator of uPAR function through complex formation with uPAR. We have identified that serum soluble LR11 (sLR11) levels are significantly elevated in patients with acute leukemia and B cell lymphomas, and are associated with tumor burden and BM invasion (Sakai et al 2012). We have also found that high sLR11 levels had a significant negative prognostic impact on progression-free survival (PFS) in FL. Therefore, we have retrospectively evaluated the clinical characteristics of sLR11 and its prognostic impact on FL, in a larger patient cohort. Patients and Methods: Sixty-one patients with FL treated at Chiba University Hospital and affiliated hospitals from 2002 to 2012 were evaluated. The majority of patients were treated by the R-CHOP regimen (rituximab 375 mg/m2 on day 1; cyclophosphamide, 750 mg/m2 on day 1; adriamycin, 50 mg/m2 on day 1; vincristine, 1.4 mg/m2 on day 1; and prednisolone, 100 mg/body on day 1–5). Serum sLR11 levels were measured by ELISA method. Patient laboratory data and treatment outcome were obtained retrospectively. Results: Serum sLR11 levels of patients with lymphoma were significantly increased (mean ± SD: 19.4 ± 17.1 ng/ml) compared with those of normal control subjects (8.8 ± 1.79 ng/ml, P 〈 0.0001). Paired sample analysis at diagnosis and at remission showed significant reduction of sLR11 levels at disease remission (22.0 ± 23.9 ng/ml vs. 8.1 ± 3.0 ng/ml, P 〈 0.0001). Multiple stepwise linear regression analysis showed that the serum sLR11 level at diagnosis was independently associated with BM invasion, lower Hb levels and elevated b2-microglobulin levels (r2= 0.48, BM invasion: P=0.0333, lower Hb levels: 0.0004, elevated b2-microglobulin levels: P=0.0289). Patients with “high” FLIPI-2 score showed significantly higher sLR11, compared to those with “intermediate” and “low” FLIPI-2 scores (29.2 ± 5.1 vs. 13.8 ± 7.7 ng/ml, p 〈 0.0001). The receiver operating characteristic curve analysis showed that the cut off level of 15.4 ng/ml provided a maximum sensitivity (0.86) and specificity (0.82) for “high” FLIPI-2 score; the area under the curve was 0.84. At the median follow-up period of 15.7 months, the probability of 5-year PFS was significantly higher in patients with sLR11 〉 15.4 ng/ml at diagnosis compared with those with ≤15.4 ng/ml (Figure 1, 9.7 % vs 100 %, P 〈 0.0001). Cox regression analysis showed that serum sLR11 levels 〉 15.4 ng/ml at diagnosis was a significant prognostic factor for PFS (hazard ratio: 2.65 × 107, 95% CI: NA). Conclusions: Serum sLR11 levels in FL patients were associated with BM invasion, lower Hb levels and elevated b2-microglobulin, which are 3 of 5 variables in FLIPI-2. This suggests that serum sLR11 is a useful tool to predict “high” FLIPI-2 score, especially highlighting those with high tumor burden by simple serum evaluation and can be a promising biomarker in patients with FL. Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V120.21.2673.2673

Language: English

Publisher: American Society of Hematology

Publication Date: 2012

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 55-55

Abstract: Introduction: Multiple Myeloma (MM) is a genetically complex and evolutionary process with well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. A small number of detectable pre-malignant clones are present in early stage and continue to acquire more genomic abnormalities leading to overt disease. The interaction between cancer cells and their environment is reciprocal, multiple components in the tissue environment can influence cancer clonal evolution and cancer cells in turn can also remodel the microenvironment and further disseminate to spatially separated areas of BM. To accurately predict the course of disease with the presence of BM environment, we require methods to estimate clone-specific growth rates and define clones that have the propensity of dissemination. Methods: We developed a novel 'bone chip' MM metastatic xenograft model using fluorescent protein tagged 'rainbow' system which enables both molecular profiling and functional tracking of clonal dissemination of tumor cells by performing tumor-bearing bone chip implantation subcutaneously to SCID-beige mice (SCID-murine model). Rainbow MM cells with equal proportion of all 15 colors were injected into donor femurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. Tumor clones in the implanted bone chip (primary sites) and distant host BM (metastatic sites) were purified by sorting and underwent RNA sequencing. By intersecting differentially expressed genes, we identified a set of genes, the expression of which were altered during disease dissemination and designated this set of genes as 'metastatic signature'. In addition, we also performed genome-wide CRISPR/Cas9-mediated loss-of-function screen in a subcutaneous xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM. The cell library infected with human sgRNA library was injected subcutaneously into SCID-Beige mice on both flanks. When metastasis was established, the fractions of each sgRNA of the primary and metastatic tumors were calculated to identify genes that facilitate tumor metastasis. Results: We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from SCID-murine model demonstrated a distinct gene expression profile of the metastatic tumors. Gene Set Enrichment Analysis of the metastatic signature in publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature is significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Through genome-wide CRISPR screening in vivo, we found that the gene targets of the most enriched sgRNAs in the BM samples were preferentially involved in important cellular processes, such as cell cycle regulation and several oncogenic signaling pathways. Additionally, many sgRNAs that remained the implanted sites until late stage were depleted during dissemination, indicating their targeted genes were important for progression. These depleted sgRNAs mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors. By using a network-based inference of protein activity method, we chose 4 genes (HMGA1, KLF6, TRIM28 and PA2G4) and validated in SCID-murine model using CRISPR mediated loss-of-function screen which prioritized HMGA1 as the key regulator in MM dissemination. Conclusions: Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. We then established a platform for future invivo CRISPR screens to investigate essential genes of response to targeted therapies and/or immunotherapies. Furthermore, a metastatic gene signature was identified and among these, HMGA1 was validated as potential regulator of MM metastasis. Disclosures Roccaro: AMGEN: Other: Advisory Board; GILEAD: Research Funding. Ghobrial:Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy; Janssen: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-113916

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 14-15

Abstract: Introduction: Recent studies based on next-generation sequencing revealed the genetic landscape of diffuse large B cell lymphoma (DLBCL). A new genetic classification system was proposed, including four groups; the BCL2 and SGK1 groups that were both associated with germinal center B-cell like (GCB) DLBCL, an MYD88 group that was associated with non-GCB DLBCL, and a NOTCH2 group that was not associated with a specific cell-of-origin (COO). Unfortunately, this classification method is somewhat complex and thus not fully accepted in clinical practice. Furthermore, part of all cases remained unclassified; as such, it is clear that the diversity of the genetic landscape of DLBCL is not yet fully understood. In this study, we performed a retrospective analysis of genetic abnormalities and their correlations with clinical characteristics in a Japanese cohort of patients diagnosed with DLBCL. Materials and Methods: We collected clinical data from 224 of a total of 234 patients who were diagnosed with DLBCL between 2013 and 2018 at Chiba University Hospital. We also collected formalin-fixed paraffin-embedded (FFPE) specimens obtained from 204 patients via standard diagnostic procedures. The COO for each sample was assessed using the Hans algorithm with immunohistochemical staining; rearrangements of BCL2, BCL6, and MYC were evaluated by fluorescence in situ hybridization. Targeted DNA sequencing was performed using a 144-gene custom panel. Results: Of the 224 DLBCL patients evaluated in our study, the median age at diagnosis was 69 years (range, 18 - 92 years). The median follow-up time was 34 months; three-year progression-free survival (PFS) was 63.2%, and three-year overall survival was 78.0%. COO was determined in 218 cases; 93 of these cases (41.5%) were classified as GCB type and 125 cases (55.8%) as non-GCB type. We successfully performed targeted DNA sequencing in 167 of the 204 FFPE samples. Compared to previous reports, our cohort included relatively higher frequencies of mutations in PIM1 (44.3%), KMT2D (40.7%), MYD88L265P (37.7%), and CD79B (24.5%); by contrast, we detected comparatively lower mutation frequencies in B2M (6.6%), ETV6 (5.4%), CDKN2A (4.7%), and TNFAIP3 (4.1%). Multivariate adjustment of genes extracted by univariate analysis and factors identified by International Prognostic Index score revealed that genetic mutations of MYD88L265P (Hazard ratio [HR] = 2.045), IL16 (HR = 4.848), and BCOR (HR = 6.295) were significant prognostic factors. Mutations in IL16 and BCOR are novel factors that will be used to predict poor prognosis. In the 167 sequenced cases, 110 were classified into the aforementioned four subtypes based on mutations detected in 23 genes together with rearrangements of BCL2 and BCL6; specifically, 47 cases were classified as members of the MYD88 group, 28 in the BCL2 group, 19 in the NOTCH2 group, and 16 in the SGK1 group. We identified a trend toward differential PFSs among the four groups (p = 0.18); three-year PFS was 51.2% among patients with MYD88 group, 59.6% in the BCL2 group, 63.2% in the NOTCH2 group, and 75.0% for the SGK1 group. When compared outcomes of patients in the BCL6 group, patients in the MYD88 group had significantly inferior PFS (p = 0.043) among the non-GCB DLBCL patients; those in the BCL2 group revealed a trend toward inferior PFS compared with those in the SGK1 group (p = 0.34) among the GCB DLBCL patients. We next focused on 28 cases of CD5-positive DLBCL; this is a distinct DLBCL subtype with poor prognosis that is identified more frequently among Asians. We observed significantly higher frequencies of frameshift or nonsense mutations in CD58 and missense mutations in MYD88L265P. Furthermore, evaluation of gene expression data for CD5-positive DLBCL reported in a published database (Miyazaki K et al. Int J Hematol. 2015) revealed diminished expression of CD58 (p = 0.01) and augmented expression of MYD88L265P (p & lt; 0.001). These results suggest that there may be a specific correlation between genetic mutations and gene expression. Intriguingly, these specific genes are key regulators of the immune response, suggesting that inflammation contribute to the unfavorable outcome of CD5-positive DLBCL. Conclusion: Our study provides support for the genetic classification of DLBCL using targeted DNA sequencing to evaluate outcomes in our Japanese cohort. We also identified mutations in novel candidate genes that may be used to predict clinical outcomes. Disclosures Nakaseko: Novartis Pharma KK: Speakers Bureau; Pfizer Japan Inc.: Speakers Bureau. Kaneda:SRL Inc.: Research Funding; SCRUM Inc.: Research Funding; Tsubakimoto Chain Co.: Research Funding; Takeda Foundation: Research Funding; Princess Takamatsu Cancer Research Fund.: Research Funding; Wedge Co Ltd: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2020-134891

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 28-29

Abstract: Introduction Despite recent developments on various transplantation procedures and supportive therapy, nonrelapse mortality (NRM) after allogeneic stem cell transplantation (allo-SCT) remains an essential issue. In choosing the appropriate regimen for allo-SCT, decision-making information that considers the complexity of different risk factors is vital. The Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI), which was initially derived and validated by investigators at the Fred Hutchinson Cancer Research Center to predict NRM, has become a widely validated tool for predicting outcomes in many transplant settings (Sorror et al. Blood. 2005). It can also stratify patients for the risk of other outcomes, including overall survival and graft versus host disease. Patients with a high HCT-CI score tend to prefer allo-SCT with reduced-intensity conditioning. Conversely, for those who prefer allo-SCT with myeloablative conditioning (MAC) and has a low HCT-CI score, a prognostic indicator is unnecessary. Furthermore, the risk factors for NRM may differ among various conditioning regimens. Therefore, the current study aimed to establish a new prognostic model for patients specific to each MAC regimen before allo-SCT. Methods We performed a retrospective cohort study to develop prognostic models of NRM in patients conditioned with cyclophosphamide/total body irradiation (Cy/TBI) or busulfan/cyclophosphamide (Bu/Cy). We selected patients who had leukemia and lymphoma in remission or had untreated or stable myelodysplastic syndrome and experienced initial allo-SCT relapse between 2007 and 2017 in the Kanto Study of Group for Cell Therapy (KSGCT). The primary outcome measure was 2-year NRM. Furthermore, we evaluated variables such as patient age, albumin, liver function, renal function, respiratory function, ejection fraction (EF), C-reactive protein (CRP), stem cell source, donor type, antithymocyte globulin use, performance status, recipient/donor sexes, time interval from diagnosis to transplant, and HCT-CI score. To identify a set of variables for Cox proportional hazards, we used an Akaike Information Criterion (AIC)-based variable selection procedure. We assigned weights to individual parameters according to their prognostic significance in Cox proportional hazard models. The identified model's discriminative ability was assessed by Harrell's C-statistic calculated using the bootstrap method. Results Among the 555 patients analyzed, 338 received Cy/TBI, and 217 received Bu/Cy. In Cy/TBI and Bu/Cy, the median age was 39 (11-60) and 44 (18-62) years, the HCT-CI score ≤ 2 was observed in 82.1% and 87.6%, and 2-year NRM was found in 13.5% and 16.0% of the patients, respectively. Before transplantation, the most dominant parameters in Cy/TBI were abnormal liver function (AST/ALT or bilirubin & gt;upper limit of normal) and albumin value & lt; 4.5g/dL, whereas those in Bu/Cy were age & gt;40 years, EF & lt; 65 %, and CRP ≥ 0.2 mg/dL. Internal validation with bootstrap resampling showed good discrimination, with C-statistic values of 0.70 (95% CI: 0.69-0.71) in Cy/TBI and 0.68 (95% CI: 0.67-0.69) in Bu/Cy. Each of the abovementioned parameters, including age & gt;40 years, was scored as 1 point. To evaluate the 2-year NRM, we divided the total scores into three risk groups. In the Cy/TBI group, the NRM was 6.9% in low (score 0-1, n = 186), 19.5% in intermediate (score 2, n = 127), and 35.3% in high (score 3, n = 25) scores. In the Bu/Cy group, the NRM was 8.3% in low (score 0-1, n = 93), 21.7% in intermediate (score 2, n = 98), and 29.8% in high (score 3, n = 26) scores (Figure). Higher scores were strongly associated with worse NRM and survival. Conclusions Our prognostic models for NRM estimation can distinguish patients with a high NRM risk. To our knowledge, these models are the first prognostic models used to estimate NRM for standard-risk patients specific to each MAC regimen. This new simple index may help predict NRM and choose an appropriate conditioning regimen before allo-SCT. Figure 1 Disclosures Nakasone: Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Janssen Pharmaceutical: Honoraria; Eisai: Honoraria; Chugai Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria. Fujisawa:Takeda Pharmaceutical Company Limited.: Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau; Novartis Pharma KK: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Janssen Pharmaceutical K.K: Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Research Funding. Nakaseko:Novartis Pharma KK: Speakers Bureau; Pfizer Japan Inc.: Speakers Bureau. Kanda:Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceuticals: Honoraria; Alexion Pharmaceuticals: Honoraria; Shire: Honoraria; Daiichi Sankyo: Honoraria; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Mochida Pharmaceutical: Honoraria; Mundipharma: Honoraria; Sanofi: Honoraria, Research Funding; Meiji Seika Kaisha: Honoraria; Shionogi: Research Funding; Otsuka: Honoraria, Research Funding; Celgene: Honoraria; Chugai Pharma: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Janssen: Honoraria; Astellas Pharma: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Pfizer: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2020-134539

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 502-502

Abstract: Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P 〈 0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P 〈 0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P 〈 0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P 〈 0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P 〈 0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.502.502

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 4483-4483

Abstract: Introduction The bone marrow (BM) microenvironment in multiple myeloma (MM) plays a pivotal role in tumor growth and bone destructive process. Mesenchymal stromal cells (MSCs) in MM exhibit different genomic and cytokine secretion profiles that ultimately impair their osteogenic differentiation abilities compared to normal MSCs. However, the underlying molecular mechanisms are not fully understood. In the present study, we explored the role of miR-138 in MSCs derived from MM patients (MM-MSCs) and the potential for anti-miR-138 treatment to rescue impaired osteogenic differentiation in MM, both in vitro and in vivo using a human xenograft MM model. Materials and methods Primary BM aspirates were obtained from MM patients and normal healthy donors, after obtaining informed consent in accordance with the Declaration of Helsinki. MiR-138 expression in MM-MSCs was measured by quantitative real-time PCR. Publicly available microarray data sets (GSE17306 and E-TABM-508) were analyzed for miR-138 expression in MM cells compared to normal plasma cells. To test the effect of inhibiting miR-138 function, a high-affinity 15-mer locked nucleic acid (LNA)-modified anti-miR oligonucleotide and a corresponding scramble sequence control oligonucleotide were used (In collaboration with Dr. Kauppinen, Denmark). Anti-miR-138 oligonucleotides were transfected into MM-MSCs or normal MSCs co-cultured with MM cell lines and osteogenic differentiation in MSCs was assessed by alizarin red staining. For the in vivo studies, 6-week-old female SCID-beige mice (n=6, each group) were injected intravenously with anti-miR-138 or scramble control oligonucleotides (15 mg/kg) 2 times a week. 3 weeks later, GFP+Luc+ MM.1S cells (3 × 106) were injected into mice. Anti-miR-138 or control oligonucleotides were continued until day 28 after injection of myeloma cells. At day 28, the effect of anti-miR138 was assessed by the number of osteoblastic lineage cell (OBC: Lin-/CD45-/CD31-/CD51+/Sca-1-) from hematopoietic cell-depleted, collagenase-treated crushed bones of mice by flow cytometry. Results MiR-138 expression in MSCs from MM patients (n=10) was significantly higher than MSCs from normal donors (n = 4) (P 〈 0.05). In addition, miR-138 expression was significantly higher in MM patient tumor cells compared to normal plasma cells using two independent data sets (GSE17306 and E-TABM-508), (P 〈 0.01 and P 〈 0.01, respectively). In three-dimensional co-culture system of MSCs from normal donors (n=6) with MM.1S cells for 2 weeks (GSE60423), miR-138 expression was increased in 4 out of 6 donors compared to MSCs cultured alone (P 〈 0.05). MM-MSCs (n≥3) transfected in vitro with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation after 3-4 weeks compared to MSCs with scramble control oligonucleotides (P 〈 0.01). Under in vitro two-dimensional co-culture conditions with MM cell lines, normal MSCs transfected with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation compared to MSCs with scramble control oligonucleotides (P 〈 0.001). In an in vivo human xenograft MM model, treatment of anti-miR-138 significantly increased the number of OBCs in the endosteal (Lin-/CD45-) BM stromal fraction of MM bearing SCID-beige mice at day 28 compared to scramble control oligonucleotides (P 〈 0.05). Conclusions These findings indicate that miR-138 plays an important role in impaired osteogenic differentiation in MSCs in MM. Inhibition of miR-138 promotes osteogenic differentiation of MSCs in MM and anti-miR-138 treatment holds the potential to prevent MM induced bone loss and lytic lesions. Additional studies are ongoing to further understand the connection between MM cells and MSCs mediated by miR-138. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Novartis: Honoraria; Noxxon: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.4483.4483

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 3430-3430

Abstract: Introduction: Citron rho-interacting serine/threonine kinase (CIT) is a serine/threonine kinase which is a key component of the midbody and is essential for cytokinesis. CIT localizes to the central spindle and midbody and functions to promote efficient cytokinesis. CIT knockdown may disrupt cytokinesis and therefore cell growth. CIT has been reported to be upregulated and important for growth of several cancers. However, the significance of CIT has not been investigated in the field of multiple myeloma (MM). We therefore dissected the role of CIT in MM growth in vitro and in vivo. Materials and methods: CIT gene expression in MM cells was compared to normal plasma cells using public-available gene expression profile (GEP) data set (GSE6477). Kaplan-Meier curve for MM patient survival between high and low CIT expressing patients were examined by using the GEP data set (GSE4581). Protein expression of CIT in MM cells was confirmed by proteomic analysis and immunohistochemistry. Knockdown of CIT was performed in MM cell lines MM1s and OPM2 using lentiviral shRNAs. CIT knockdown was confirmed by reduced CIT mRNA in comparison to a scrambled control. Differences in cell proliferation and cell cycle between CIT knockdown cells and scramble control were analyzed by using thymidine uptake and PI staining, respectively. Cytokinesis failure was analyzed by immunofluorescence using alpha-tubulin antibody and DAPI. shCIT OPM2 (n=7) and the scrambled control cells (n=8) were injected subcutaneously into SCID-Bg mice (5x106 cells/mouse) and were followed for tumor development and survival. Results: CIT expression was significantly higher in MM patients’ plasma cells compared to healthy donors in GEP (p=0.02), proteomic analysis and immunohistochemistry. Also CIT expression was higher in relapsed patients compared to newly diagnosed patients by GEP. MM patients with high CIT expression had significantly worse overall survival compared to low CIT expressing patients (p=0.04). CIT knockdown MM cell lines showed reduced cell proliferation and G2 cell cycle arrest by thymidine uptake and PI staining compared to the scrambled control. Significantly, large amount of multinucleated cells, which indicates cytokinesis failure, were observed in the CIT knockdown cells compared to scrambled control. Reduced tumor growth (p 〈 0.001) and prolonged survival (p 〈 0.001) was observed in CIT knockdown MM cell line injected mice. Conclusions: shRNA knockdown of CIT in MM cells induces G2 arrest leading to cytokinesis failure in vitro with reduced cell proliferation in vivo. Since MM cells have significantly higher expression of CIT compared to normal plasma cells, CIT represents a novel therapeutic target for MM. Studies are ongoing to develop drugs to target CIT for MM treatment. Disclosures Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.3430.3430

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1793-1793

Abstract: Platelets play important roles in both physiological and pathological conditions. For instance, platelets may facilitate cancer metastasis by protecting circulating tumor cells (CTCs) from shear stress and immunological assault during their intravascular phase and by supporting CTCs extravasation. Moreover, soluble factors released from activated platelets enhance proliferation and migration of endothelial cells, thereby promoting tumor angiogenesis. Correlations between increased platelet counts and shorter survival time have been described for many solid tumors. However, the role of platelets/megakaryocytes (MKs) in regulating tumor progression and dissemination in Multiple myeloma (MM) has not been previously examined. We measured the platelet aggregation-inducing abilities of MM cell lines and found MM cell lines (MM.1S, OPM-2, KMS-11, U266, and H929) induced platelet aggregation. This was not observed using leukemia cells (K562) and primary healthy donor-derived peripheral blood mononuclear cells. We next investigated whether platelets interact with MM cells within the bone marrow (BM) niche, in vivo. In order to identify platelets/MKs, femurs were harvested from MM.1S GFP+-harboring mice and stained with a DyLight649-conjugated anti-GPIb-beta antibody. The femurs were rendered transparent by the CUBIC method as previously described (Cell 159, 911-24 (2014)) and examined using confocal microscopy; and found that platelets/MKs co-localized within MM.1S GFP+-infiltrated BM niches. Further confirmation of the co-localization of MM cells and MKs was performed using immunohistochemistry (CD138+ and GPIb-alpha+). We next investigated the effect of platelets on MM cell proliferation: MM cells, where co-cultured with platelets and found that platelets enhanced the proliferation rate of MM cells in a platelet number-dependent manner, as shown by using BrdU (p 〈 0.001). The observed platelet-dependent induction of MM cells proliferation was also demonstrated by using platelet aggregation releasate. We next performed RNA sequencing studies using MM cells and platelet-interacting MM cells, and demonstrated that platelet-interacting MM cells presented with enrichment for signaling pathways such as TGF-beta as compared to MM cell control. We performed cytokine array analysis and confirmed that several cytokines, including TGF-beta, ENA-78, SDF-1, and TARC, were released from platelets activated by co-culture with MM cells. We next investigated the effect of platelets on MM tumor growth in vivo, by using bioluminescence imaging (BLI). MM cells were pre-cultured with platelets for 2 days, washed and then intravenously injected into SCID/Beige mice (n=6). We found a significantly higher MM tumor burden in mice injected with MM cell pre-cultured with platelets, as compared to control mice where buffer treated MM cells were used (p 〈 0.01), together with reduced survival (p=0.0011). These findings indicate the ability of platelets to enhance MM cell growth in vivo. To further confirm the role of platelets in mediating MM tumor growth, we performed platelet depletion by treating mice with an anti-platelet antibody (R300, 4 ug/g IP injection), and subsequently injected MM cells intravenously. We found that platelet-depleted mice presented with decreased MM tumor growth (p 〈 0.05) and improved survival rate compared to control mice treated with IgG isotype control (p=0.0044). These findings indicate that MM cells induce platelet aggregation and interact with platelets/MKs within the BM. Importantly, MM cell-platelet interaction is responsible for the activation of pro-survival signaling pathways in MM cells at transcriptome level, leading to enhanced MM cell proliferation, as shown both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.1793.1793

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Leukemia & Lymphoma, Informa UK Limited, Vol. 61, No. 1 ( 2020-01-02), p. 221-224

Type of Medium: Online Resource

ISSN: 1042-8194 , 1029-2403

URL: Article

DOI: 10.1080/10428194.2019.1660973

Language: English

Publisher: Informa UK Limited

Publication Date: 2020

detail.hit.zdb_id: 2030637-4

In: Leukemia Research Reports, Elsevier BV, Vol. 2, No. 2 ( 2013), p. 67-69

Type of Medium: Online Resource

ISSN: 2213-0489

URL: Article

DOI: 10.1016/j.lrr.2013.07.002

Language: English

Publisher: Elsevier BV

Publication Date: 2013

detail.hit.zdb_id: 2706248-X