ALBERT

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Key Points CYR61/CCN1 is a bone marrow microenvironmental biomarker for myeloma progression and for transformation of MGUS and asymptomatic disease to overt myeloma. CCN1 reduces myeloma bone disease and tumor growth and is a potential therapeutic target for myeloma.

Description: Abstract 5014 Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival. To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation. These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Jumping translocations of 1q12 (JT1q12) provide a mechanism for the deletion of 17p in cytogenetically defined high-risk myeloma. Sequential JT1q12s introduce unexpected copy number gains and losses in receptor chromosomes during subclonal evolution.

Description: Abstract 3717 Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by 〉37% (p

Description: Inhibition of integrins αvβ3 and αvβ5 in human brain microvascular endothelial cells (HBMECs) by the function-blocking peptide RGDfV induces loss of spreading on vitronectin, cell detachment, and apoptosis. We demonstrate that cell detachment is not required for apoptosis because plating on bovine serum albumin–blocked poly-L-lysine (allows attachment, but not integrin ligation and cell spreading) also induced apoptosis. Latrunculin B (LatB), which inhibits F-actin polymerization, induced transient loss of HBMEC spreading on vitronectin, but not their detachment, and induced apoptosis despite recovery of cell spreading. However, LatB did not cause apoptosis in 5 tumor cell lines. In HBMECs, both LatB and RGDfV induced transient Y412 and Y245 phosphorylation of endogenous c-Abl, a nonreceptor tyrosine kinase that reciprocally regulates F-actin. LatB also induced nuclear translocation of c-Abl in HBMECs. STI-571 (imatinib), a targeted therapy for BCR-ABL1+ leukemias and inhibitor of c-Abl, platelet-derived growth factor receptor, and c-Kit, decreased endothelial apoptosis. LatB-induced HBMEC apoptosis, and its inhibition by STI-571 also occurred in a 3-dimensional collagen model, supporting physiologic relevance. Last, siRNA to c-Abl (but not nonspecific siRNA) also inhibited RGDfV- and LatB-induced apoptosis. Thus, endogenous c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3/αvβ5 or by LatB-induced disruption of F-actin.

Description: Abstract 808 Background: Bone disease is one of the most debilitating complications in patients with multiple myeloma (MM). The molecular mechanisms by which MM triggers bone disease are not fully understood. We have previously demonstrated that Dkk1 is highly expressed in primary MM plasma cells, and associated with bone disease in MM patients by inhibiting Wnt signaling-promoted mesenchymal stem cell differentiation and osteoprotegerin production in osteoblast cells. We have also reported that increase in Wnt signaling in the bone marrow microenvironment by overexpression of Wnt3a in myeloma cells or administration of rWnt3a, or indirectly increasing Wnt signaling by administration of anti-Dkk1 neutralizing antibody also decreased in osteoclast numbers. However, Dkk1 is less frequently expressed in MM cell lines that are derived mostly from late stage of MM; and injection of these MM cell lines into human fetal bone also is able to induce bone lesion in MM animal model. These results indicate that additional factors may be involved in induction of the bone disease at the stage of the disease. The members of the sFRPs family of secreted proteins (including sFRP-1, -2, -3 and -4) directly bind to Wnts, thereby preventing Wnts from binding to the cellular Wnt receptor complex. It has also been reported that sFRP-1 and -2 augment canonical Wnt3a activated signaling in fibroblast. MM cells from pateints with advanced bone lesions express sFRP2 mRNA. Like sFRP2, sFRP3 mRNA is highly expressed in MM plasma cells, but it's function in MM bone disease remains unknown. We sought to investigate the role of sFRP3 in MM-triggered bone lesions using the osteoblast (OB) cell lines CH3T1/2 and C2C12, and serum from MM pateints those MM cells expressed high level of sFRP3. Methods/Results: RT-PCR analysis showed that sFRP3 is expressed in primary MM plasma cells and certain MM cell lines. Recombinant sFRP3 protein did not inhibit, but synergized with Wnt3a to increase beta-catenin protein, while Dkk1 significantly inhibited this process. Similarly, sFRP3 treatment of OB cells increase Wnt-3a-induced TCF transcript activity in OB cells transfected with TOPflash luciferase report constructs. sFRP3 also increased MSC differentiation, as evidenced by increase in alkaline phosphatase activity (ALP) and increased in mineralization by Alizarin red staining. sFRP3 treatment also increases OPG mRNA and protein production in these cells. Similar to sFRP3, sFRP1 and sFRP2 synergistically acted with Wnt3a to induce MSC differentiation and OPG expression in osteoblasts, while Dkk1 significantly inhibited these processes. To confirm the synergistic effects of sFRPs with canonical Wnt signaling on MSC differentiation, we employed R-podin1, a well-known agonist of canonical Wnt signaling. Treatments of MSC cells with R-podin1 led to increase in beta-catenin protein and TCF transcriptional activity and in ALP activity, and increase in OPG mRNA and protein. Pretreatment of the cells with sFRP2 and sFPP3 proteins further enhanced the function of R-podin1. In contrast, Dkk1 protein showed negative effect on R-Spodin1 functions, indicating that sFRP2 and sFRP3 synergized with R-Spodin1 to induce activation of canonical Wnt signaling and subsequent MSC differentiation and OPG production. Conclusion: Taken together, these data suggest that sFRP2 and sFRP3 augment canonical Wnt signaling to induce MSC differentiation and indirectly inhibit osteoclastogenesis by regulating OPG in MSC cells. These results also indicate that Dkk1 may be most important in MM-induced bone disease. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Description: Abstract 4603 Introduction: Successful transplantation of cryopreserved hematopoietic stem cell (HSC) can be regularly achieved provided sufficient numbers of cells are administered. However the optimal conditions for preparation, freezing and thawing remain to be defined. The duration of hematopoietic stem cell viability is unclear. The ultimate measure of viability has remained in vivo hematologic recovery following transplantation. Evidence of autologous repopulation in preclinical setting has been seen at 14 years after bone marrow transplant and after 12 years in the clinical setting after peripheral stem cell transplant. We report a successful transplantation in a patient in whom autologous cryopreserved marrow with cellular dose of 1.21 × 108/kg was infused 21 years following collection. Case: The patient is 43 year old man found to have follicular lymphoma with bone marrow involvement in 1989 at age 22. He achieved complete remission after treatment with two cycles of Chorambucil. Bone marrow procurement and cryopreservation was performed at that time for possible subsequent infusion. The procured marrow consisted of a total cell count of 1.21 × 10^8 cells/per kg body wt with a total volume of 354 ml. Equal parts of 20% DMSO were combined with marrow to a final concentration of 10% DMSO. The marrow was then stored in the liquid phase of nitrogen from date of collection until date of infusion 21 years later. Our patient relapsed in 1996, and eventually underwent treatment in 2006 with six cycles of Fludarabine and Rituximab, achieving a complete remission. He continued Rituximab therapy maintenance every 6 months for a total of 2 years. During Rituximab therapy, he was noted to have pancytopenia Work-up confirmed MDS with 5q- and translocation of long arm of chromosome 6q21 and short arm 17p13 in 20/20 cells by karyotype analysis. Patient elected to proceed with the cryopreserved marrow transplant. Assessment of cryopreserved marrow for dysplasia was undertaken showing no evidence of cytogenetic or histological changes. The patient was prepared with Busulfan IV at 0.8/kg q 6 hours × 4 days and Cyclophosphamide 60mg/kg IV × 2 days. The marrow was infused 21 years following its procurement. Samples from the infused marrow showed 65–75% viability by Tryptan blue assay. White cell engraftment occurred on day 17 and platelet reached 20,000 by day 30. Follow-up 2 months post transplant revealed persistent mild pancytopenia, WBC of 2.6 with ANC 1500, Hgb 9.8 and platelets of 43,000. FISH analysis for 5q performed showed 85/200 cells positive for 5q-. Bone marrow biopsy confirmed dysplastic features consistent with his pre-transplant bone marrow biopsy. Our case illustrates that even in the setting of marginal numbers of infused marrow components and after prolonged cryopreservation, repopulation can readily occur. However, inability to eliminate the malignant clone following stable engraftment brings to light both the necessity of adequate ablation in pretransplant conditioning, and the importance of graft-versus-tumor effect in marrow born malignancies. To our knowledge, this is the oldest successful cryopreserved autologous bone transplant at 21 years post preservation. As novel uses of stem cells advance, optimal storage of various cellular components is necessary. This should be further investigated on a larger scale in the future. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2957 Myeloma is intimately associated with osteolytic bone disease, resulting from myeloma cells' interactions with osteoclasts and osteoblasts and their progenitors, and is dependent on the changes it induces in bone metabolism for progression. Myeloma cell dependence on the bone marrow microenvironment is also evident experimentally, where interaction of primary myeloma plasma cells (MMPC) with osteoclasts (OC) and with mesenchymal stem cells (MSC) support the survival of primary myeloma cells. To understand the molecular mechanisms associated with the survival of MMPC, we used Acuity 4 software to analyze Affymetrix U133 Plus2 chip data and identify changes in gene expression in induced MMPC freshly isolated from 8 patients by interaction with OC and from 8 additional patients with MSC. Expression by MMPC of 675 genes was changed following interaction with OC; 552 genes were upregulated and 123 down regulated. Expression of 296 genes was changed in MSC co cultures (161 upregulated, 135 down regulated). Comparison of the genes whose expression was similarly changed in both co culture systems identified 72 probesets, representing 58 genes, that were commonly changed; 33 were upregulated and 25 down regulated. Ingenuity Pathway Analysis assigned 54 of the 58 genes to 4 distinguished networks of interrelated genes with high probability scores. We next tested the hypothesis that the expression of genes whose expression was commonly changed in the co culture systems has clinical significance. To accomplish this, we used gene expression data available on 127 relapsed patients who had been uniformly treated on our Total Therapy 2 protocol, and for whom gene expression (GEP) data at first relapse (RL) were available. 71 of these patients also had pre treatment (BL) GEP data; for these 71 patients we calculated change in expression of the 72 probesets as the ratio of RL/BL expression signal. We identified 7 genes whose expression changes were significantly (p≤0.05) associated with survival after relapse: These genes were, in order of significance: CCNE2, PECAM1, KLHL21, ICAM1, PLAU, ANPEP, and DUSP1, with p-values ranging from 0.017 to 0.05. Up regulation of PECAM1, ANPEP, DUSP1, and down regulation of CCNE2, KLHL21, ICAM1, and PLAU were associated with longer survival. We further determined whether expression level of these 72 probesets at relapse, defined by signal intensity, correlated with post relapse survival of the 127 patients; 18 genes were significantly (p

Description: Frommeyer, Epstein and Taylor: Refractoriness in hemophilia to coagulation-promoting agents: Whole blood and plasma derivatives. Blood 5: 401-420 (May), 1950. The authors wish to note that the following changes should have appeared in the article: Page 402, first line: "Lawrence and Johnson, 1941,8 reported" (instead of "Lawrence and Johnston, in 1946,8 reported"). Page 402, second sentence of same paragraph: "In this instance the patient had received whole blood and plasma in therapeutic management prior to the appearance of resistance to therapy." (instead of ". . . had received chemically prepared plasma fractions in the form of Fraction I of Cohn in addition to whole blood and plasma in therapeutic management prior to . . . "). Page 420, reference 8. To this reference should be added: "(Presented before the American Clinical and Climatological Society, October 1941.)".