ALBERT

Description: The identification of novel tumor-associated antigens, especially those shared among patients, is urgently needed to improve the efficacy of immunotherapy for multiple myeloma (MM). In this study, we examined whether Dickkopf-1 (DKK1), a protein that is not expressed in most normal tissues but is expressed by tumor cells from almost all patients with myeloma, could be a good candidate. We identified and synthesized DKK1 peptides for human leukocyte antigen (HLA)–A*0201 and confirmed their immunogenicity by in vivo immunization in HLA-A*0201 transgenic mice. We detected, using peptidetetramers, low frequencies of DKK1 peptide-specific CD8-positive (CD8+) T cells in patients with myeloma and generated peptide-specific T-cell lines and clones from HLA-A*0201-positive (HLA-A*0201+) blood donors and patients with myeloma. These T cells efficiently lysed peptide-pulsed but not unpulsed T2 or autologous dendritic cells, DKK1-positive (DKK1+)/HLA-A*0201+ myeloma cell lines U266 and IM-9, and, more importantly, HLA-A*0201+ primary myeloma cells from patients. No killing was observed on DKK1+/HLA-A*0201-negative (HLA-A*0201−) myeloma cell lines and primary myeloma cells or HLA-A*0201+ normal lymphocytes, including B cells. These results indicate that these T cells were potent cytotoxic T cells and recognized DKK1 peptides naturally presented by myeloma cells in the context of HLA-A*0201 molecules. Hence, our study identifies DKK1 as a potentially important antigen for immunotherapy in MM.

Description: Abstract 2060 Background: Three subtypes of MPN (myelofibrosis (MF), polycythemia vera (PV) and essential thrombocythemia (ET)) are characterized by activation of JAK2 signaling, with the majority having the JAK2 V617F mutation. Median survival in these subtypes ranges from months to years for MF (Gangat JCO 2011) and up to a decade or more for some patients with PV and ET. Some symptomatic treatment options exist, but with the exception of hematopoietic stem cell transplant, none are curative. Little is known about healthcare costs associated with these diseases. Objective: To compare annual medical and pharmacy costs in three subtypes of MPN patients (MF, PV, ET) to matched non-cancer controls using a large US commercial claims database. Methods: Data were drawn from the Thomson Reuters MarketScan database, which contains claims for insured employees, retirees and dependents from approximately 100 US payers. Index date was the first claim with an MPN diagnosis code in the time period 2005 to 2008. Eligible patients had one year continuous enrollment post-index with no evidence of non-AML secondary malignancy. Controls had one year of continuous enrollment after their match date and were matched to patients in a ratio of 5:1 based on gender, year of birth, geographic region and insurance type (Comprehensive, Health Maintenance Organization, Preferred Provider Organization, etc.). Controls were excluded if they had a service claim with a diagnosis for cancer or a pharmacy claim for chemotherapy. Costs were based on total gross payments to the provider, which equals the amount eligible for payment under the medical plan terms after applying rules for discounts, but before applying coordination of benefits, copayments and deductibles. Total cost was the sum of MPN and non-MPN related medical costs which included inpatient, outpatient and emergency room service and pharmacy. Pharmacy included injectable and non-injectable chemotherapy, supportive care and other prescription drug costs. Results: A total of 25,145 MPN patients (MF: 509, PV: 16,165, ET: 8,471) were included and assigned matching controls. The mean cost (standard deviation) for cases and controls are shown in the table. Medical and Pharmacy Costs: Conclusions: Annual medical and pharmaceutical costs for patients with MPNs are 2 to 6 times those of matched non-cancer patients and represent medical management challenges and payer burden. Outpatient visits accounted for about half of the total healthcare costs, followed by inpatient visits, pharmaceutical costs and ER visits. Disclosures: Price: Elil Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Pohl:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Xie:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Walgren:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership.

Description: Background Existing data demonstrate that SREs can impose a significant economic burden. Much of the cost data for MM patients are combined with other tumor types and do not make comparisons between MM patients with SRE and MM patients without SRE. This research provides updated, comprehensive cost data in MM patients to address these gaps. Methods Patients 18 years of age or older were required to have ≥2 claims in any position with a diagnosis of MM (ICD-9-CM codes 203.00, 203.01, 203.02), at least 30 days apart, between 01 January 2005 and 31 December 2010; the date of the first MM diagnosis was the index date. Marketscan(r) data were used to select patients continuously enrolled in a non-capitated commercial health plan with a medical and pharmacy benefit for 12 months before the index date (i.e., baseline period) and at least 3 months after the index date (post-index period). Patients were followed until the earliest of death or disenrollment from the health plan or end of the study period on 31 December 2011. MM patients with ≥ 1 SRE were compared to those without SRE to characterize the associated incremental cost of SRE. Frequency of SREs, HCRU (events/person-mth), and costs (USD/person-mth) were determined. Cost is defined as total gross payment to providers for a specific service, which includes amount paid by the patient and payer. Due to skewed distributions, bootstrapping (replication=1000) methodology was used to estimate standard error of rates of HCRU and costs and to compare cohorts. P-values were generated utilizing 2-sample t-tests. Results MM patients with SRE (n=596, mean age=67.8, 45.6 % male, mean Charlson/Deyo [CD] =1.1) were compared to MM patients without SRE (n=432, mean age=65.7, 47.5 % male, mean CD=0.8). Patients are categorized by number of SREs experienced during the study period in Table 1. MM patients with SRE experienced significantly greater HCRU during both the baseline and post-index periods (Tables 2&3). The HCRU increases translated into mean total costs for patients with SRE that were significantly greater during both the baseline (w/o SRE: USD 953; w/SRE: USD 1328; p=0.005) and post-index (w/o SRE: USD 3307; w/SRE: USD 4763; p

Description: To develop more effective immunotherapy strategies for patients with multiple myeloma (MM), it is important to identify novel tumor antigens. Heat shock protein gp96, a highly conserved glycoprotein, is a chaperon molecule that carries intracellularly processed peptides from proteins synthesized by the cells, including those of tumor-specific or -associated proteins. Recent studies in solid tumors have shown that tumor-derived gp96 is immunogenic and potent at stimulating the generation of tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we examined whether myeloma-derived gp96 can be used as potent myeloma antigen. To generate myeloma-specific CTLs, immature dendritic cells (DCs), obtained from cultures of blood monocytes from HLA-A2+ healthy individuals or patients, were pulsed with gp96 protein purified from the myeloma cell line U266 (A2+), and induced maturation using the cytokine cocktail. Autologous T cells were then stimulated every two weeks with these DCs, and cytotoxicity was examined against gp96-pusled DCs, myeloma cell lines, and primary myeloma cells isolated from patients. After several cycles of in vitro stimulation, specific CTL lines were obtained, which consisted of both CD4+ and CD8+ T cells. These CTLs demonstrated not only specific cytotoxicity against gp96-pusled DCs and the cell line U266, but also significantly killed A2+ primary myeloma cells. No killing was observed against A2+ normal lymphocytes including B cells or A2− myeloma cell lines and primary myeloma cells from patients. Using the cold target inhibition assay, we showed that the same CTLs mediated the killing of both gp96-pulsed DCs and myeloma tumor cells. The cytotoxicity was MHC class I- and, to a lesser extent, class II-restricted, indicating that the gp96 contained both class I- and II-restricted tumor-derived peptides and that myeloma cells naturally present these peptides in the context of their surface MHC molecules. Upon antigen stimulation, these CTLs secreted predominantly interferon-g, detected by the ELISPOT assay and intracellular staining, indicating that they belong to the type-1 T-cell subsets. Furthermore, the CTLs lysed the target cells mainly through the perforin-mediated pathway because Concanamycin A but not Brefeldin-A, the selective inhibitors for perforin- or Fas-, respectively, mediated pathways, abrogated the cytolytic activity of the cells. These results therefore show that myeloma-derived gp96-specific CTLs are able to lyse myeloma tumor cells including primary myeloma plasma cells from patients and, thus, provide a rationale for gp96-based immunotherapy in MM.

Description: Multiple myeloma (MM) is a B-cell malignancy often associated with a suppressed immune system. The mechanisms for the immunosuppression are largely unknown. In this study, we examined, using the murine 5T33 myeloma model, the effects of tumor cell or its-derived factors on the differentiation and function of bone marrow-derived dendritic cells (BMDCs). Our results showed that differentiation of BMDCs was retarded in the presence of 10% of tumor-derived supernatant (TSN). This is evident by, compared with control cells, the downregulated expression of surface molecules including CD40, CD86 and MHC class II; secretion of higher levels of IL-10 and lower levels of IL-12; and a poor T-cell response in an allogeneic mixed lymphocyte reaction induced by TSN-treated cells. The same phenomenon was also observed when the bone marrow progenitor cells were cocultured, either in direct contact or separated by transwell membrane, with myeloma cells. The treatment downregulated the expression of phosphorylated extracellular signal-related kinase (ERK) and mitogen-induced extracellular kinase (MEK), and upregulated the expression of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription-3 (STAT3) in the cells. As a high level of interleukin (IL)-10, IL-6, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-b can be detected in TSN, we examined whether these cytokines were responsible. Our results showed that addition of TGF-b, IL-10 and/or IL-6 could largely replace TSN in retarding the differentiation of BMDCs, and neutralizing antibodies against these cytokines, especially in combination, were able to block the effects of TSN or tumor cells on BMDCs. Finally, our results showed that inhibiting p38 MAPK or STAT3 restored the differentiation and function of these cells. Hence, our study not only sheds light on the mechanisms of tumor-induced immune evasion in MM and but also provides one of the solutions to overcome the problems.

Description: Abstract 3140 Background: Three subtypes of MPN (myelofibrosis (MF), polycythemia vera (PV) and essential thrombocythemia (ET)) are characterized by activation of JAK2 signaling, with the majority having the JAK2 V617F mutation. Patients diagnosed with MF, PV or ET are typically at higher risk for infections, cardiovascular complications, anemia and other comorbid diseases (Barosi Blood 2007, Vannucchi Blood 2007). However, comparative rates of these comorbidities are not well described in the literature. Objective: To compare comorbidity rates from three subtypes of MPN patients (MF, PV, ET) with matched non-cancer control groups using a large US commercial claims database. Methods: Data were drawn from Thomson Reuters MarketScan, which contains claims for insured employees, retirees, and dependents from approximately 100 US payers. Index date was the first claim with a diagnosis code for MPN in the time period 2005 to 2008. Eligible patients had 1 year of continuous enrollment post-index with no evidence of non-AML secondary malignancy. Patients were deemed to have a comorbidity if they had an ICD-9 diagnosis code that was on a list of pre-specified codes likely to be related to MPN for each comorbidity category. Controls had one year of continuous enrollment after their match date and were matched to patients in a ratio of 5 :1 based on gender, year of birth, geographic region and insurance type (Comprehensive, Health Maintenance Organization, Preferred Provider Organization, etc.). Controls were excluded if they had a service claim with diagnosis for cancer or a pharmacy claim for chemotherapy. Results: A total of 25, 145 MPN patients were included and assigned matching controls. For MF, PV and ET cases, respectively, mean age was 64.4, 54.4. 52.3 years and % female was 51.7, 36.1 and 70.7. Comorbidity rates during the study period for some diseases of interest in MPN patients and matched controls are shown in the table. Conclusion: Patients diagnosed with an MPN typically experienced significantly higher rates of comorbidities than non-MPN matched controls. These findings have implications for both the clinical management of MPN patients as well as for health economic assessments, since a substantial portion of the cost of care for MPN patients may reside in treatment of comorbidities not directly coded to MPN. Restricting patients and controls in this study to a single matching year of observation helped to align risk between cases and controls, but may have underestimated the total comorbidity burden that MPN patients could experience in their lifetime. Disclosures: Price: Elil Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Pohl:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Xie:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Walgren:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership.

Description: Two common features in human immunodeficiency virus infection and acquired immunodeficiency syndrome, rheumatoid arthritis, and hematologic malignancies including multiple myeloma are elevated serum levels of β2-microglobulin (β2M) and activation or inhibition of the immune system. We hypothesized that β2M at high concentrations may have a negative impact on the immune system. In this study, we examined the effects of β2M on monocyte-derived dendritic cells (MoDCs). The addition of β2M (more than 10 μg/mL) to the cultures reduced cell yield, inhibited the up-regulation of surface expression of human histocompatibility leukocyte antigen (HLA)–ABC, CD1a, and CD80, diminished their ability to activate T cells, and compromised generation of the type-1 T-cell response induced in allogeneic mixed-lymphocyte reaction. Compared with control MoDCs, β2M-treated cells produced more interleukin-6 (IL-6), IL-8, and IL-10. β2M-treated cells expressed significantly fewer surface CD83, HLA-ABC, costimulatory molecules, and adhesion molecules and were less potent at stimulating allospecific T cells after an additional 48-hour culture in the presence of tumor necrosis factor-α and IL-1β. During cell culture, β2M down-regulated the expression of phosphorylated mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), and mitogen-induced extracellular kinase (MEK), inhibited nuclear factor-κB (NF-κB), and activated signal transducer and activator of transcription-3 (STAT3) in treated cells, all of which are involved in cell differentiation and proliferation. Thus, our study demonstrates that β2M at high concentrations retards the generation of MoDCs, which may involve down-regulation of major histocompatibility complex class I molecules, inactivation of Raf/MEK/ERK cascade and NF-κB, and activation of STAT3, and it merits further study to elucidate the underlying mechanisms.