Citations

Abstract Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

Abstract Background: Patients with SMM/IMM are at high risk for progression to active MM and are appropriate candidates for chemoprevention trials. High IL-1beta levels are a useful surrogate marker for progression from SMM/IMM to active myeloma. Methods: We carried out a Phase II clinical trial of IL-1Ra (Anakinra) in patients with SMM/IMM to determine the biologic activity, progression-free rate, and toxicity of IL-1Ra. Since IL-1beta induces paracrine IL-6, we hypothesized that IL-1Ra would inhibit IL-6 production and myeloma cell growth. Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Non-progressors were allowed to continue on therapy with IL-1Ra until they converted to active myeloma. IL-1beta levels were measured in a bioassay with a read out of IL-1 inducible IL-6 production. CRP levels served as a surrogate for IL-6 production. Results: Thirty-six patients are included for analysis based on intent to treat at diagnosis. At baseline, twenty-nine patients had elevated functional IL-1beta levels consistent with progressive disease and 12 patients had radiologic evidence of disease on bone survey. The median TTP for the entire group was 1 year and 2 patients exhibited a minor response based on M-protein reduction. A proportional hazards analysis was performed and the variables examined were: % bone marrow plasma cells, plasma cell labeling index (PCLI), circulating plasma cells, albumin, M-protein level, IgA subtype, CRP, CRP % reduction ≥ one-third, beta-2 microglobulin, creatinine, calcium, hemoglobin, urine total protein, presence of lytic bone disease, and IL-1 level. Univariate Cox model results showed that % BMPC, PCLI, circulating plasma cells, CRP % reduction, presence of lytic disease, and IL-1 level were all statistically significant (p < .05) variables. However, only the CRP % reduction from baseline remained significant in the multivariate analysis (p < 0.01). The median TTP for patients without (n=18) and with (n=18) a decrease in CRP was 6 months and > 2 years, respectively (p < .0001). Toxicities included injection site reactions during the first month of therapy and asymptomatic neutropenia necessitating a reduction in therapy to every other day. Five patients in whom the CRP decreased and the baseline PCLI was > 0 also demonstrated a parallel decrease in the PCLI. Several of the patients with progressive disease on IL-1Ra alone could later be rescued with the addition of low dose dexamethasone (20 mg once a week) suggesting that the endogenous IL-1beta levels were too high to inhibit with IL-1Ra alone. Conclusion: In summary, IL-1Ra can prolong the TTP to active myeloma in responsive SMM/IMM patients through inhibition of IL-6 production (decrease in CRP) and myeloma cell growth. Figure Figure

Abstract Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.

Abstract Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo. Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension. Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3. Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.

Abstract Background: It has been hypothesized that multiple myeloma (MM) remains incurable due to a stemcell/proliferative component that responds only partially to standard treatment regimens. We have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6, the central myeloma growth factor, and that IL-1Ra will inhibit paracrine IL-6 production in vitro. Based on these preclinical studies, we have developed a Phase II trial using IL-1Ra. Purpose: To determine the biologic and clinical activity of IL-1Ra (Anakinra) in a subgroup of smoldering MM (SMM) patients that present with an elevated plasma cell labeling index (PCLI; a measure of myeloma cell proliferation). Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Results: Eleven of 29 patients enrolled on the protocol had an on-study PCLI > 0 and have completed six months of therapy with IL-1Ra. Seven of the 11 patients had a decrease in the PCLI of 75–100% (see Table), three had only modest changes (≤ 50%), and one had an increase. The decrease in the PCLI paralleled a decrease in the high sensitivity C-reactive protein (CRP) in all cases (43–90% decrease). The above results suggested that IL-1Ra inhibited IL-6 production in the myeloma microenvironment, as evidenced by a reduction in the CRP, resulting in suppression of myeloma cell growth. However, there was little effect on the M-protein. To investigate these clinical observations, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone (DEX), IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed: 1) IL-1Ra was superior at inhibition of IL-6 but caused no increase in apoptosis; 2) the DEX apoptotic effect was eliminated by IL-6 3) DEX and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. Based on these in vitro results, 5 of the 11 patients have been advanced to IL-1Ra + low dose DEX (20 mg/week) resulting in 4/5 minor responses (25–50% decrease M-spike) and 1/5 with stable disease. Conclusion: The use of IL-1Ra is a novel targeted therapeutic strategy that interferes with myeloma cell growth. By inhibiting IL-1beta induced IL-6 production, IL-1Ra specifically targets the proliferative myeloma fraction and also complements DEX induced apoptosis. Preliminary studies on the use of IL-1 inhibitors in SMM patients to delay/prevent progression to active MM and to minimize toxicity appear encouraging, however, more patients need to be studied. Patient # On Study 6 months % Change 1 PCLI 0.8% 0% −100% CRP (mg/L) 2.01 0.58 − 71% M-spike (g/dL) 2.5 2.4 − 4% 2 PCLI 0.2% 0% − 100% CRP 0.89 0.51 − 43% M-spike 2.8 2.7 − 3% 3 PCLI 0.2% 0% − 100% CRP 5.7 1.02 − 82% M-spike 2.9 2.9 0% 4 PCLI 4.1% 1% − 75% CRP 3.39 1.51 − 55% M-spike 3.8 3.6 − 5% 5 PCLI 0.4% 0% − 100% CRP 3.46 0.34 − 90% M-spike 2.7 3.1 15% 6 PCLI 0.3% 0% − 100% CRP 17 5.35 − 68% M-spike 4.4 3.9 − 11% 7 PCLI 1.2% 0% − 100% CRP 1.06 0.32 − 70% M-spike 3.1 2.9 − 6%

8590 Background: Merkel Cell Carcinoma (MCC) is an aggressive cutaneous malignancy of neuroendocrine origin associated with immunosuppression presumably through infection with Merkel Cell Polyomavirus present in >80% of patients (pts). The impact of immune status on clinical outcomes in pts with MCC is unknown. The primary objective of this study was to compare clinical characteristics and outcomes of pts with MCC who are immunosuppressed (ISP) versus non-immunosuppressed (non-ISP). Methods: We performed a retrospective chart review on pts with MCC diagnosed at the Mayo Clinic between 1981 and 2009. A dermatopathologist confirmed all cases. ISP was defined as pts diagnosed with chronic lymphocytic leukemia, HIV, solid organ transplant recipients, or chronic immunosuppressive medication. The association between ISP status and overall survival was summarized using the hazard ratio (HR) and 95% confidence interval (CI) estimated from a Cox regression model. Results: Of the 268 pts identified and included in the study, 38 (14%) were ISP. We found no differences in age, tumor size, tumor location, stage of disease, or recurrence rate in ISP vs Non-ISP. Among pts who had Stage 3-4 disease, there was no difference in the size of the primary between groups. Among pts with Stage 1-2 disease, ISP status was not significantly associated with poorer survival (HR 1.5, 95% CI 0.7-3.3). However, among pts with Stage 3-4 disease, ISP pts had significantly poorer survival compared to non-ISP pts (HR 2.7, 95% CI 1.2 - 6.2). Conclusions: Baseline clinical characteristics of pts with MCC do not differ based on immunosuppression, and outcomes do not differ in pts in regards to immunosuppression in early stage MCC (Stage 1-2). However, in pts with Stage III-IV MCC, ISP pts have a worse clinical outcome suggesting that metastatic MCC either behaves more aggressively in ISP pts either through intrinsic differences in the biology of the tumor or improved immune evasion in ISP pts. These results should be cautiously interpreted given the small number (n=12) of immunosuppressed pts with advanced stage MCC. The authors will update their data with an additional 58 patients by the date of presentation.

8105 Background: IL-6 is the central myeloma growth factor and we have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6. A Phase II trial was completed using IL-1ra, which inhibits paracrine IL-6 production, and low dose Dexamethasone (Dex), which decreases IL-1 levels through myeloma cell apoptosis, in patients with smoldering/indolent MM (SMM/IMM). These patients are at the greatest risk for progression to active MM and most likely to benefit from anti- cytokine therapy. Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. A total of 47 patients received 100 mg of IL-1ra SQ qd for 6 months unless clinical progression occurred. Responding patients were allowed to continue on therapy with IL-1ra alone. Low dose Dex (20 mg qweek) was added after 6 months of IL-1ra in non-responding patients. The primary endpoint was progression-free survival (PFS). Results: The 47 patients were at high risk for progression to active MM with 98% having ≥ 10% plasma cells, 89% generating IL-1 levels consistent with myeloma (≥ 1.0), and 32% having bone lesions. All 47 patients received IL-1ra initially and 25/47 subsequently received IL-1Ra/Dex. For the group of 47 patients, the median overall progression-free survival was 37.5 months. Three patients achieved a minor response (MR) to Anakinra alone; 5 pts achieved a PR and 4 patients an MR after addition of Dex. Seven patients had a decrease in the plasma cell labeling index (PCLI; a marker of myeloma cell growth) on Anakinra alone which paralleled a decrease in the high sensitivity CRP (a marker of serum IL-6 levels). The median PFS for patients without (n=12) and with (n=35) a ≥ 15% decrease in their baseline hsCRP was 6 months and > 3 yrs, respectively (p=0.002). Conclusions: In SMM/IMM patients at high risk for progression to active myeloma, treatment with IL-1 inhibitors (IL-1ra ± Dex) results in an increased PFS in patients that demonstrate a ≥15% reduction in the baseline CRP compared to those that do not respond. No significant financial relationships to disclose.

Abstract We have evaluated the efficacy of a novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor, ITF2357, to promote cell death in multiple myeloma (MM) cells. HDAC inhibitors, which promote histone hyperacetylation and increase gene expression, have been evaluated as candidate agents for combating malignancies because they impact the expression of genes related to proliferation, differentiation, and survival. Exposure of MM cell lines to 1 micromolar ITF2357 led to dramatically increased levels of histone acetylation at 4 hours and 8 hours by Western analysis. Sub-micromolar concentrations of ITF2357 promoted time- and concentration-dependent cell death in MM cell lines. Using 500 nM ITF2357, a concentration potentially achievable in vivo, viability of KAS-6/1 IL-6 dependent myeloma cells was reduced to 28% of control at 24 hrs and 2% of control at 48 hours (Figure 1). In contrast, viability of normal PBMCs was 100% at 24 hours and 80% at 48 hours (Figure 2). U266 and 8226 myeloma cells were found to be sensitive to ITF-2357 in a similar fashion with U266 being least sensitive. Cell death proceeded via apoptosis as measured using Annexin V/propidium iodide staining. ITF 2357 was superior to suberoylanilide hydroxamic acid (SAHA) at inhibition of stromal cell IL-6 production. IL-1beta (10 pg/ml) was used to stimulate bone marrow stromal cell IL-6 production (105 ng/ml) after 48 hours. Concentration of ITF2357:Stromal Cell IL-6 production after 48 hours were as follows - 10 nM: 78 ng/ml; 100 nM: 79 ng/ml; 1000 nM; 32 ng/ml. SAHA at similar concentrations showed no significant decrease in stromal cell IL-6 production compared with the no drug control. In summary, ITF2357 induces significant myeloma cell apoptosis and can inhibit stromal cell IL-6 production. It represents an attractive therapeutic candidate for MM clinical trials. Figure Figure Figure Figure

Abstract We investigated whether interleukin-1β (IL-1β) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1β appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1β was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1β mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1β message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1β mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1β production. In the future, continued follow-up of IL-1β positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1β is predictive of those MGUS patients that will eventually progress to active myeloma.