Progression Of MM Research Articles

Hypermethylation Is A Key Feature of the Transition of Multiple Myeloma to Plasma Cell Leukemia

AbstractAbstract 535PCL is associated with a failure of the plasma cells to retain their normal homing patterns to the bone marrow, is difficult to treat and developing targeted treatments based on its pathogenesis would be a step forward. PCL lies at the end of the multistep pathway from normal to malignant plasma cells and little is known about the genetic mechanisms mediating the final stages of this pathway. The methylation status of genes in myeloma can change as the malignancy progresses and as such identifying genes deregulated by methylation that mediate the progression of MM to PCL may offer epigenetically relevant therapeutic targets. We have previously analyzed 181 samples including B cell, normal plasma cells, MGUS and MM samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Here we have added 32 PCL samples to our dataset in order to further investigate the methylation changes that occur on the transition from MM to PCL. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Data were analyzed in GenomeStudio using the methylation module (Illumina). Further analyses were performed using R and the LIMMA package. Differential methylation between samples was identified using an empirical Bayes moderated t-test and the resulting p-values were adjusted using the Benjamini and Hochberg method. P-values below 0.05 were considered significant. We have shown genome-wide hypomethylation at the transition from MGUS to MM with gene-specific hypermethylation of tumor suppressor genes. The methylation patterns within myeloma samples correlate with translocations or hyperdiploidy, where the latter are split into 2 distinct clusters with a significant difference in overall survival, p=0.03. However, the t(4;14) subgroup had the most distinct methylation profile with hypermethylation of genes specific to the t(4;14) subgroup including CD79A, FAM49A and SOCS2 which correlate with loss of expression in this subgroup. Herein, we analyzed presentation MM (n=161) and PCL (n=32) samples for methylation changes which may contribute to expression and phenotypic changes. When comparing all MM and PCL samples, irrespective of cytogenetic subgroup, we find hypermethylation of the genome in PCL samples. Upon clustering we find that PCL samples segregate within the translocation groups with which they belong i.e. t(11;14) PCL samples cluster with t(11;14) myeloma samples. However, when MM and PCL samples from the same cytogenetic group are compared there is exclusive hypermethylation of genes occurring as the disease progresses. Although t(4;14) myeloma samples have considerable more methylation than t(11;14) samples, the increase in methylation from MM to PCL is consistent in both groups, resulting in t(4;14) PCL samples with increased methylation over t(11;14) PCL samples. The genes which are hypermethylated in the transition of MM to PCL can be classified as either tumor suppressor genes, genes involved in cell-cell signaling, or as cell adhesion molecules. The further analysis of these genes will allow us to identify genes which are down-regulated through methylation and mediate the progression of MM to PCL allowing the clone to become independent of the bone marrow microenvironment. In addition, these analyses will enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. In conclusion, we have demonstrated that methylation changes play a significant part in differentiating the various cytogenetic subgroups of MM and in mediating the transition to PCL. Hypermethylation affects genes and pathways important in retaining plasma cells in the bone marrow as well as in their growth factor independent growth in the absence of stromal cell support. Disclosures:No relevant conflicts of interest to declare.

In Vivo Imaging Model of Multiple Myeloma and Its Cellular Interaction with the Bone Marrow Microenvironment

BACKGROUND: Imaging animal models that offer serial measurement of systemic tumor progression, such as the GFP+ or bioluminescence MM model, have been limited to low resolution, gross measurements of tumor progression that are insufficient to detect individual cells, and their interaction with their microenvironment. Therefore, the need exists for development of sensitive, high resolution three-dimensional imaging methods that identify the dynamic changes that occur during tumor initiation and progression. We here show the use of in vivo fluorescence confocal microscopy to follow MM tumor initiation and progression at the cellular level using stably GFP-transfected MM1S cells in a xenograft model of MM.METHODS: 5 × 10 6 MM1S-GFP-Luc cells were injected into the tail veins of non-irradiated SCID/Beige male mice. MM cell growth in the marrow of the calvarial bone was analyzed using in vivo flow cytometry and fluorescence confocal microscopy, as previously described (Sipkins et al 2005). High-resolution images with unprecedented cellular detail were obtained through the intact mouse skull at depths of up to 250μm. To visualize the bone marrow vasculature the mice were injected with a blood pool marker (Angiosense 680 or 750) immediately before imaging, and to delineate the surface of calvarial bone, a fluorescent hydroxyapatite tag (Osteosense) was used. The validity of the imaging data was established by sacrificing select mice, and analyzing the previously imaged tissues by standard histologic and immunohistologic techniques. After MM tumors became established in the fourth week following injection, 1 mg/kg Bortezomib was administered twice weekly to a subset of the mice, these were imaged following treatment along with controls that were not treated. For all mice imaged, the number and areas of the skull where GFP+ MM cells were found were recorded. Confirmation of homing and tumor progression was also performed using CD138+ selected primary tumor cells.RESULTS: Using this model, we were able to detect and monitor individual GFP+MM cells within the bone marrow microenvironment. We demonstrate that MM.1 S cells and primary CD138+ cells exit the systemic circulation within one hour of injection, followed by specific rolling and adhesion to the vasculature of the bone marrow microenvironment. Within 4 days post after injection, the MM cells were fully engrafted along the bone marrow sinusoids, which were surrounded by bisphosphonate-rich bone structures including ostoeoblasts. Within the second week, loose clusters of a few cells began to form around the blood vessels. Growth and expansion appeared to be closely associated with the vasculature. Tumor growth dramatically increased in the third week following cell injection when areas of the parasagittal regions became completely involved with MM cells. In contrast, standard bioluminescence imaging performed concurrently detected tumor initiation only at 4 weeks post-injection, indicating that confocal microscopy is a much more sensitive technique in detecting early tumor proliferation. Imaging of bortezomib-treated mice demonstrated that tumor size and density was reduced in the skull, but even more dramatically the number of sites containing GFP+MM was greatly reduced.CONCLUSIONS: Our imaging model differs from other models due to its unprecedented resolution. Therefore it is particularly useful for following small numbers of tumor cells either early in disease progression or after therapeutic treatment. This model offers a more sensitive spatial and temporal live imaging of MM cells in the BM microenvironment and can be used to explore the dynamic interaction of MM with different structures and environments of the BM. We anticipate that this model will allow for a better understanding of the biologic effects of therapeutic agents on the growth of MM cells within the bone marrow niches.

Gene expression signatures of conventional prognostic factors in multiple myeloma

6606 Background: Recent advances in Multiple Myeloma have led to identification of novel therapeutic targets and new agents highlighting the need for patient specific treatment plans. We have initiated molecular studies of MM plasma cells (PC) to correlate conventional prognostic factors with the genomic signature of MM PCs. Factors with high discriminatory value will allow clinicians to tailor therapy to the patient, decreasing toxicity and improving outcome. Methods: Gene expression profiling was performed on CD138 sorted PCs from 14 pts with newly diagnosed MM using the Affymetrix U133A platform. The data was analyzed using dChip software (V1.3). Results: We have identified differentially expressed genes with biological relevance that correlates with the important prognostic factors; plasma cell labeling index (PCLI), circulating PCs, LDH and β2 microglobulin. High PCLI was associated with upregulation of growth and proliferation related genes (IGFBP-7, WHSC1 [mapped to 4p16 and correlated with t(4;14)], TIMP1 [related with STAT3 phosphorylation] and down regulation of wnt5a, CD79a (MB1, member of Ig superfamily gene), NEDD9 (associated with β1 integrin signaling), BCR1 (B cell receptor signaling) and Lysosomal associated multispanning membrane protein 5 (described in MM progression). Increased number of circulating PCs was associated with modulation of adhesion-related and migration associated genes such as CD86, metastasis associated protein S100A4, galectin-1 (associated with β1 integrin), hsp27, glucosamine-6-sulfatase, NCAM1, and cyclin D1, among others. Similar analyses performed grouping patients according to LDH, β2 microglobulin also revealed differentially expressed genes that may have correlative significance.Conclusions: Identification of differentially expressed genes that correlates with MM-related prognostic factors can help understand the biological basis for these factors. Additionally, identification of non-overlapping discriminatory genes for individual prognostic factors can lead to development of focused arrays that can be used clinically for prognostication. Ongoing studies with additional patients as well as in vitro studies are underway to validate these results. No significant financial relationships to disclose.

Cytokines Behaviour in Multiple Myeloma during Zoledronic Acid Treatment.

The main pathological feature underlying the bone disease in multiple myeloma is an uncoupling of bone resorption from bone formation so that resorption predominates. In the bone marrow (BM), MM plasmacells are in close contact with stromal elements and induce the release of several cytokines which modify the BM microenviroment, upregulating RANKL expression and secretion by both stroma and osteoblasts. Destruction of bone matrix induced by MM and its cellular interactions is accompanied by further release of cytokines. Bisphosphonates are chemical compounds which selectively concentrate at the interface of the active osteoclasts and the bone resorption surface where they inhibit osteoclast activity.Recently, in solid tumors, Santini et al have demostred that zoledronic acid could have an in vivo antiangiogenic property through a significant and long-lasting reduction in serum VEGF levels (Clin Cancer Res 2003; 9:2893–7). Taking into account that angiogenesis plays an important role in MM progression, we have investigated the behaviour of IL6, PDGF, IGF, TNFα and VEGF in a cohort of 28 (16 males and 13 females) consecutive MM pts with lytic bone lesions treated with 4 mg of zoledronic acid. Venous blood samples were drawn just before the beginning of drug infusion and at 1,2,7,21 (before the subsequent infusion) days after the zoledronic acid infusion. Serum samples were then tested for PDGF, VEGF, IL6, TNFα , and IGF with the R & D quantitative kits (R & D Systems, Minneapolis, USA), according to the manufacturer's instructions. Basal cytokine levels were compared with the values observed at 1, 2, 7 and 21 days using the Wilcoxon's test for nonparametric-dependent continuous variables. Differently from what observed by Santini et al in solid tumors, serum VEGF median levels did not decrease;on the contrary, it significantly increased at days 7 (P=0.0047). Moreover, IL 6 and TNFα levels significantly increased after 1 (P=0.0000 and 0.0001 respectively) and 2, ( P=0.0000 and 0.0021 respectively) days. Furthermore, PDGF significantly decreased (P=0.005) after 2 days following zoledronic acid infusion. Among these pts, 8 (28.5%) were receiving also Interferon; no statistically significant differences in the levels of VEGF, IL6, TNFa and IGF between the two groups were observed. Only the PDGF levels have shown a statistically significant decrease in the interferon group at 0, 1, 2, 7, 21 days (P<0.05). In conclusion, the cytokines' behaviour after the administration of zoledronic acid in MM is different from that observed in solid tumors; in particular, we did not observe a decrease in VEGF levels. This behaviour may partially explain why zoledronic acid has no effect on MM progression as originally postulated.

Bone Marrow Angiogenesis and Progression in Multiple Myeloma: Clinical Significance and Therapeutic Approach

It is now well established that solid tumors depend on angiogenesis. Promoters and inhibitors of angiogenesis are in balance and antiangiogenic strategies aim at repressing the angiogenic process, thus retarding solid tumor progression. Recent data suggest the importance of angiogenesis in hematologic malignancies and several studies reveal an increased angiogenesis in active multiple myeloma. Angiogenesis seems to be a prominent feature of MM progression, and seems to be correlated with the prognosis and the resistance of MM to chemotherapy. Numerous cell populations and cytokines are involved in angiogenesis in multiple myeloma and antiangiogenic therapy with thalidomide is effective in patients with refractory or relapsed disease. The combination of thalidomide and of other immunomodulatory agents with other therapeutic regimens could lead to more effective management of patients with multiple myeloma.