ALBERT

Description: Introduction The incidence of multiple myeloma (MM) increases with age, yet some cytogenetic changes are actually more common in younger patients with MM (Avet-Loiseau J Clin Oncol 2013).  This suggests that a mechanism other than chromosomal changes underlies the increased incidence with age.  Senescent cells secrete a number of proinflammatory cytokines, chemokines, growth factors and proteases resulting in the senescence-associated secretory phenotype (SASP), which can promote tumor growth.  Preclinical data suggests that myeloma bone marrow stromal cells express the SASP (Andre PLOS ONE 2013). We hypothesized that SASP factors correlate with age in patients with MM. Methods Peripheral blood serum and matched bone marrow aspirate plasma from patients with multiple myeloma were evaluated for selected factors associated with the SASP using quantitative multiplex immunoassay (Rules Based Medicine, Austin TX USA).  SASP factors with a known role in MM [interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15), granulocyte-macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1(ICAM1), osteoprotegerin (OPG), hepatocyte growth factor (HGF), insulin-like growth factor-binding protein(IGFBP-1), interleukin-1 beta (IL1b), monocyte chemotactic protein 1(MCP-1), macrophage inflammatory protein-1 alpha(MIP-1a), angiogenin, leptin,  vascular endothelial growth factor receptor 1(VEGFR1) and stem cell factor(SCF)] were selected. The relationship between age and SASP factors were analyzed using Kendall tau rank correlation coefficient. Results Samples from 25 patients (each with peripheral blood serum and matched bone marrow aspirate plasma) were analyzed.  The median age was 62 (range 47 - 74). Disease states were as follows: 36% newly diagnosed/untreated, 24% pretransplant and 40% relapsed.  ISS stage included 40% stage I, 28% stage II and 32% stage III.  Three of the selected SASP factors in the peripheral blood correlated   with age:  IL-8 (Kendall Tau 0.334, p=0.027), OPG (Kendall Tau 0.289, p=0.046) and MCP-1 (Kendall Tau 0.332, p=0.022).  No SASP factors tested in the bone marrow plasma were significantly correlated with age. Conclusions We demonstrated age-associated differences in the SASP factors IL-8, OPG and MCP-1 in the peripheral blood of myeloma patients.  Future research will examine differences between patients with myeloma and age-matched controls without cancer. Disclosures: Vij: Celgene : Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau. Stockerl-Goldstein:Celgene : Speakers Bureau; Celgene : Speakers Bureau; Millennium: Speakers Bureau; Millennium: Speakers Bureau.

Description: Abstract 5016 Activating mutations in Ras (N- and K-) are the most common mutations in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. As previously reported, we re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found the most common mutations to affect Ras oncogenes (N and K-). To test the role of Ras activation in MM pathogenesis directly, we generated mice harboring a mutant K-ras allele, Lox-stop-lox(LSL)- K-ras G12D (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre), activated specifically in germinal center (GC) cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12), phenotypes that have previously been reported for K-ras+/G12D mice. Tumor cells demonstrated successful recombination of the K-Ras locus. We showed recombination in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). No evidence of plasma cell proliferation was seen by serum ELISA, SPEP, flow cytometry or histological examination. We confirmed these results by crossing the K-ras+/G12D mice to a second strain, AID-Cre, which expresses the recombinase earlier and more specifically in the GC. We found 100% (n=20) of K-ras+/G12D/AID-Cre-YFP mice possessed visible, small benign papillomas on the ventral neck, as early as 3 weeks of age. To stimulate malignant transformation, vitamin D deficient chow and/or sub-lethal radiation was given to K-ras+/G12D/AID-Cre-YFP and negative control, K-ras+/G12D mice. Dual treatment increased the size and number of papillomas, giving a median survival of 26 weeks, compared to 52 weeks with control. However, no evidence of MM was detected by serum ELISA, SPEP, flow cytometry or histological examination. Finally, we generated AID-Cre-K-ras+/G12D triple transgenic mice in a tumor prone Arf-null background. After 13 wks, we found sarcomas in 66% (n=3) of K-ras+/G12D/AID-Cre-YFP/Arf−/− mice. Additionally, 100% (n=3) of these mice had rapid growing benign papillomas and only a minimal increase in the plasma cell compartment, compared to K-ras+/G12D/AID-Cre/Arf +/− control. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center or plasma cells, even in an Arf-null context, and suggests that specific myeloma events, yet to be identified, are required for malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4002 African-Americans (AA) have a 2–3-fold higher risk of multiple myeloma (MM) relative to Whites (Gebregziabher, 2006). We have formed a consortium and are conducting a multi-center study with 9 clinical centers and 4 NCI Surveillance, Epidemiology and End-Results (SEER) Program population-based cancer registries to determine the causes of the disease in this population and explain the excess risk (Myeloma in African-American Patients, MAP). Participation involves providing a blood or saliva specimen for DNA and answering a lifestyle and medical history questionnaire. At the end of the data collection period, a genome-wide scan will be performed and our results compared to those from 2,000 African-American controls participating in cohort studies. Patients with African ancestry (predominantly African-Americans) are identified from outpatient clinic rosters or from population-based cancer registries. For patients recruited at clinics, information on subtype, cytogenetics, FISH and lytic bone lesions is abstracted from medical records. To date, 601 patients have agreed to be in the study and we have received DNA samples from 592 patients; 54.6% are female and 45.4% are male. The mean age at diagnosis is 57 years (SD =11.2) with a median age at diagnosis of 58 years (range 27 to 90 years of age). Of the 514 subjects who completed a questionnaire, 7.8% were obese at age 20 (body mass index 〉 30) and 39% were obese 5 years prior to diagnosis. A first-degree relative with MM was reported by 17 cases (3%), 74% higher than the lifetime risk of 1.7% in the general population based on SEER data. In addition, cases reported 21 first-degree relatives with leukemia (4%), 7 with non-Hodgkin lymphoma (1%) and 14 with Hodgkin lymphoma (3%). To date, clinical information has been abstracted for 351 patients. Of these, 207 (58%) have active disease with the following distribution: stage I (30%), stage II (27%) and stage III (43%). The remainder have relapsed (13%), refractory (1%), relapsed and refractory (4%), or smoldering myeloma (6%), or are in remission (18%). The subtype distribution is: IgG (74%), IgA (11.4%), IgD (0.9%) and IgM (0.3%), and light chain only (13.5%); a distribution significantly different from that observed in a predominantly White population (P

Description: Primitive hematopoietic progenitors from some patients with Philadelphia chromosome (Ph)–positive chronic myeloid leukemia (CML) express aberrant transcripts for interleukin 3 (IL-3) and granulocyte colony-stimulating factor (G-CSF), and exhibit autonomous proliferation in serum-free cultures that is inhibited by anti–IL-3 and anti–IL-3 receptor antibodies. Expression of the product of the Ph chromosome, the BCR/ABL oncogene, in mice by retroviral bone marrow transduction and transplantation induces CML-like leukemia, and some leukemic mice have increased circulating IL-3, and perhaps granulocyte-macrophage colony-stimulating factor (GM-CSF). These observations raise the possibility of autocrine or paracrine cytokine production in the pathogenesis of human CML. Mice with homozygous inactivation of the Il-3 gene, the Gm-csf gene, or both, were used to test the requirement for these cytokines for induction of CML-like disease by BCR/ABL. Neither IL-3 nor GM-CSF was required in donor, recipient, or both for induction of CML-like leukemia by p210 BCR/ABL. Use of novel mice deficient in both IL-3 and GM-CSF demonstrated that the lack of effect on leukemogenesis was not due to redundancy between these hematopoietic growth factors. Analysis of cytokine levels in leukemic mice where either donor or recipient was Il-3−/−indicated that the increased IL-3 originated from the recipient, suggestive of a host reaction to the disease. These results demonstrate that IL-3 and GM-CSF are not required for BCR/ABL-induced CML-like leukemia in mice and suggest that autocrine production of IL-3 does not play a role in established chronic phase CML in humans.

Description: The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

Description: Background Recent practice guideline changes suggest all patients with MM should receive bisphosphonates, regardless of the presence of bone disease. Nitrogen-containing bisphosphonates, ZA and PAM, inhibit protein prenylation, a process crucial for osteoclast survival. This inhibition also interferes with tumor cell adhesion, angiogenesis, and proliferation suggesting potential for anti-tumor activity of bisphosphonates. Morgan et al showed increased OS with use of ZA versus clodronate in MM. This increase was independent of the effect on bone disease, supporting anti-tumor activity of ZA. ZA and PAM are the two approved bisphosphonates for MM in the United States. In in-vitro studies, ZA has increased potency compared to PAM, a finding that may result in increased apoptosis of tumor cells. A meta-analysis by Mhaskar et al demonstrated an association between higher bisphosphonate potency and improved OS in MM. In an effort to clarify the effect of ZA versus PAM on OS in MM, we evaluated outcomes in a large cohort of United States Veterans with MM. Methods We identified 1,018 patients with newly diagnosed MM in the Veterans Administration Cancer Registry between 2002 and 2009, who were treated with either PAM or ZA, but not both. Data was collected on age, co-morbidities, date of diagnosis and death, myeloma specific and supportive medications, and baseline lab data. Cox proportional-hazards was used to assess association between bisphosphonate use and OS while controlling for other prognostic factors. Propensity score analyses were performed to reduce confounding by indication, using the inverse probability weighting (IPW) approach of Cole and Hernan and propensity score matching. The covariates for each propensity model were the same as those in the main analysis. Results Of the 1,018 patients in the cohort, 383 received ZA and 635 received PAM. The median follow-up was 26.9 months. After adjustment using propensity score groups, baseline characteristics were well balanced between the groups (Table 1). Kaplan-Meier curves showing OS of patients receiving ZA versus PAM are shown in Figure 1. After controlling for age, weight, comorbidity score, era of diagnosis (before or after 2006), baseline lab characteristics, and treatment, OS was significantly improved with ZA compare to PAM (HR 0.84; 95% CI, 0.72-0.98). In both the IPW and propensity score matching analyses, ZA significantly improved OS in MM (HR 0.84; 95% CI, 0.72-0.97) and (HR 0.83; 95% CI, 0.69-0.99) respectively. Conclusion In this large, multicenter cohort study, ZA improved OS compared to PAM in patients with MM. The benefit persisted even after controlling for known patient and treatment related prognostic factors, as well as controlling for differences in baseline patient characteristics. Bisphosphonates play a key role in the treatment of MM for the prevention of MM bone disease. Evidence suggests that bisphosphonates may also have a direct anti-tumor effect in MM. Our study adds to this growing body of evidence and provides rationale for selecting ZA over PAM in most patients with MM. Disclosures: No relevant conflicts of interest to declare.

Description: The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

Description: Monoallelic chromosome 13 deletion detected by cytogenetics predicts poor patient survival in multiple myeloma (MM), but the genes responsible have not been conclusively identified. To this end, we performed array comparative genomic hybridization (aCGH) using a novel, human chromosome 13 oligonucleotide array (Nimblegen) with 385,272 probes and median probe spacing of 60 base pairs. The dense coverage, and the use of germline DNA collected from each patient as internal controls for DNA copy number polymorphisms, enabled unprecedented map resolution of somatic DNA gains and losses on chromosome 13. Array CGH was performed on genomic DNA isolated from CD138+ bone marrow plasma cells purified from 20 patients with MM, monoclonal gammopathy of undetermined significance (MGUS), or amyloidosis. Visual analysis of the aCGH data identified 4 patients with chromosome 13 interstitial deletions that were confirmed using a circular binary segment algorithm (Nimblegen). Monosomy chromosome 13 was detected in 5 patients by cytogenetics, and as expected, appeared normal by aCGH due to data normalization. We also performed an unsupervised analysis of the data, which identified 49 genes with DNA copy number decreases. Both methods identified copy number decreases at 13q14 commonly affected in MM and MGUS patients and thought to harbor a relevant tumor suppressor. Three of the 4 patients with interstitial deletions at 13q14 had striking regions of DNA copy loss whose minimally deleted region was defined by a patient with a small deletion spanning exon 20 of RB1, encoding part of the functionally important ‘pocket domain’ responsible for binding E2F transcription factors. We found RB1 protein levels in MM cell lines correlated with RB1 genomic copy number, and therefore considered the model that RB1 haploinsufficiency contributes to MM. However, we found Rb1 heterozygous (HET) and wild type (WT) mice had indistinguishable steady-state B, T and myeloid compartments in addition to plasma cell induction in response to sheep red blood cell stimulation. Disease burden was similar in HET vs. WT Rb1 mice in a model of NRAS induced tumorigenesis. These results suggest other genomic events cooperate with RB1 copy number loss in MM. Unexpectedly, we found the 3 patients that had an interstitial deletion of RB1 at 13q14 concomitantly harbored a separate interstitial loss at 13q13. Every patient with DNA copy number loss of RB1 also had DNA copy loss within 13q13 (5 patients who lost the entire chromosome and 3 patients with interstitial deletions). The minimally deleted region at 13q13 mapped to the 5′ end of Neurobeachin (NBEA), which encodes a Protein Kinase A (PKA) anchoring protein. We detected NBEA transcripts at low levels in normal human plasma cells. NBEA transcripts and protein were robustly expressed in 3/5 MM cell lines. This is the first report of coordinate copy number loss of RB1 and NBEA on chromosome 13 in MM. Taken together, our data suggest that chromosome 13 deletions in MM may target protein dose level of RB1 and at least one other gene, likely NBEA. Our data provide a novel rationale for future studies to examine the biological consequences of coordinate loss of NBEA and RB1.

Description: Ectopic expression of c-Myc (Myc) in most primary cell types results in programmed cell death, and malignant transformation cannot occur without additional mutations that block apoptosis. The development of Myc-induced lymphoid tumors has been well studied and supports this model. Myc can be upregulated in acute myeloid leukemia (AML), but its exact role in myeloid leukemogenesis is unclear. To study its role in AML, we used a murine stem cell virus (MSCV) retroviral gene transfer/transplantation system to broadly express Myc in the bone marrow of mice either alone or in combination with antiapoptotic mutations. Myc expression in the context either of Arf/Ink4a loss or Bcl-2 coexpression induced a mixture of acute myeloid and acute lymphoid leukemias (AML+ALL). In the absence of antiapoptotic mutations however, all mice transplanted with MSCV-Myc (100%, n = 110) developed AML exclusively. MSCV-Myc-induced AML was polyclonal, readily transplantable, possessed an intact Arf-p53 pathway, and did not display cytogenetic abnormalities by spectral karyotyping (SKY) analysis. Lastly, we found that Myc preferentially stimulated the growth of myeloid progenitor cells in methylcellulose. These data provide the first direct evidence that Myc is a critical downstream effector of myeloid leukemogenesis and suggest that myeloid progenitors are intrinsically resistant to Myc-induced apoptosis. (Blood. 2005;106: 2452-2461)