Kathleen A. Donovan

Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.

Induction of a Chronic Disease State in Patients With Smoldering or Indolent Multiple Myeloma by Targeting Interleukin 1β-Induced Interleukin 6 Production and the Myeloma Proliferative Component

OBJECTIVE To conduct in vitro studies as well as a phase 2 clinical trial in patients with smoldering or indolent multiple myeloma to determine if interleukin 1 (IL-1) inhibitors can delay or prevent active myeloma. PATIENTS AND METHODS Stromal cells were cocultured with IL-1beta-expressing myeloma cells in the presence of dexamethasone, IL-1 receptor antagonist (IL-1Ra), or both. Levels of interleukin 6 (IL-6) and of apoptosis were also quantified. Between November 19, 2002, and May 24, 2007, 47 patients were enrolled in the study and subsequently treated with IL-1Ra. In 25 (53%) of the 47 study patients, low-dose dexamethasone (20 mg/wk) was added. The primary end point was progression-free survival (PFS). RESULTS In vitro, IL-1Ra was superior to dexamethasone at inhibiting IL-6 production; maximal IL-6 inhibition and apoptosis induction were achieved by addition of both IL-1Ra and dexamethasone. In the clinical trial, 3 patients achieved a minor response to IL-1Ra alone; 5 patients achieved a partial response and 4 patients a minor response after addition of dexamethasone. Seven patients showed a decrease in the plasma cell labeling index that paralleled a decrease in high-sensitivity C-reactive protein (hs-CRP) levels. The median overall PFS was 37.5 months. The median PFS for patients without (n=12) or with (n=35) a greater than 15% decrease in 6-month vs baseline hs-CRP levels was 6 months and more than 3 years, respectively (P=.002). Disease stability was maintained in 8 patients who received therapy for more than 4 years. CONCLUSION In patients with smoldering or indolent multiple myeloma who were at risk of progression to active myeloma, treatment with IL-1 inhibitors decreased the myeloma proliferative rate and hs-CRP levels in those who responded, leading to a chronic disease state and an improved PFS. TRIAL REGISTRATION clinicaltrials.gov identifier: NCT00635154.

THE ROLE OF INTERLEUKIN-1β IN THE PATHOGENESIS OF MULTIPLE MYELOMA

Multiple myeloma (MM) is recognized clinically by the proliferation of malignant plasma cells in the bone marrow, the detection of a serum or urine monoclonal protein, anemia, hypercalcemia, renal insufficiency, and lytic bone lesions. 32 Monoclonal gammopathy of undetermined significance (MGUS) is characterized by a monoclonal protein (M-protein) in the serum or urine without other clinical features of multiple myeloma. 20,32 Patients with MGUS are asymptomatic and have stable serum M-protein measurements. 30 Monoclonal gammopathy of undetermined significance is more common than myeloma, occurring in 1% of the population over age 50 and 3% over age 70. 20 It is of great clinical importance to distinguish between patients with MM and those with MGUS, because MGUS patients may be safely observed off chemotherapy. Unnecessary treatment can lead to acute leukemia 20 or to morbidity or mortality from chemotherapy. Patients with MGUS usually have less than 10% marrow plasma cells, a serum monoclonal protein level of less than 3 g/dL, no urinary Bence Jones protein, and no anemia, renal failure, lytic bone lesions, or hypercalcemia. In contrast, patients with active myeloma will present with a marrow plasmacytosis of 10% or more, a serum monoclonal protein of 3 g/dL or more, a 24-hour urine monoclonal protein of 1 g or more, and lytic bone lesions. Patients with smoldering multiple myeloma (SMM) have a marrow plasmacytosis of 10% or more or a serum monoclonal protein level of 3 g/dL or more, but they have stable disease and no lytic bone lesions. Many patients with MM have a history of prior MGUS. In one Mayo Clinic study, 58% had prior MGUS or plasmacytoma. 31 During long-term follow-up of 241 patients with MGUS, 59 patients (24.5%) later developed MM or a related plasma cell proliferative disorder. 30 Closer examination of patients who developed MM or a related plasmaproliferative disorder revealed that the majority of patients remained stable for an extended period of time but subsequently progressed over a relatively short period to myeloma or a related plasmaproliferative disorder. Of the 59 patients whose disease progressed, 39 developed multiple myeloma, and 18 of these had serial serum studies performed (Table 1). In these 18 patients, the M-protein remained stable for a median of 8 years and then increased slowly over 1 to 4 years, or rapidly in less than 1 year, with the development of myeloma. 30 Based on these clinical observations, it is likely that differences exist between MGUS and myeloma and that additional changes arise in the monoclonal plasma cells leading to overt myeloma. These changes could lead to aberrant expression of cytokines, adhesion molecules, or other cellular factors that may be responsible for the transition from MGUS to SMM to active MM.

Contrast in cytokine expression between patients with monoclonal gammopathy of undetermined significance or multiple myeloma.

We investigated whether differences in IL-6 and IL-1β expression could be detected in monoclonal plasma cells from patients with MGUS or MM. Expression of IL-6 and IL-1β in bone marrow cells was determined using cell sorting to enrich for plasma cells followed by reverse transcriptase/polymerase chain reaction (RT/PCR). Nineteen patients (six MGUS, two primary amyloid (AL), 11 MM) were studied. IL-6 mRNA expression was detectable in the sorted CD38+/CD45− plasma cell populations from 0/6 MGUS, 0/2 AL and 5/11 MM patients. All five MM patients with autocrine IL-6 expression demonstrated an elevated plasma cell labeling index. IL-1β mRNA was detectable in the sorted CD38+/CD45− plasma cell populations from 1/6 MGUS, 0/2 AL and 10/11 MM patients. In situ hybridization (ISH) confirmed that the IL-1β producing cells were plasma cells. In conclusion, autocrine production of IL-6 parallels a high labeling index and aberrant expression of IL-1β correlates with the diagnosis of MM. Follow-up of IL-1β-positive MGUS patients will determine whether aberrant expression of IL-1β will predict those MGUS patients that will eventually progress to MM.

IL-1β expression in IgM monoclonal gammopathy and its relationship to multiple myeloma

We have shown that IL-1β is not detectable in normal plasma cells but is produced by plasma cells from virtually all patients with multiple myeloma (MM). To extend our earlier work, IL-1β expression was determined in 13 newly diagnosed patients with IgM monoclonal gammopathy. Eleven patients with Waldenstroms macroglobulinemia (WM) and two patients with IgM MM were investigated for IL-1β expression by in situ hybridization (ISH). All patients with WM had bone marrow biopsies consistent with the diagnosis, an IgM M-protein in the serum, and subsequently required chemotherapy. Seven of 11 patients with WM had an M-protein >3 g/dl and five patients had bone surveys performed that were negative for osteolytic disease. Two patients were diagnosed with IgM MM because of the presence of significant osteolytic disease on a metastatic bone survey. ISH for kappa, lambda, and IL-1β expression was performed on bone marrow aspirates from each of the 13 patients. None of the neoplastic cells from the 11 patients with WM showed detectable IL-1β expression by ISH. However, the neoplastic cells from both patients with IgM MM expressed IL-1β mRNA at high levels. This aberrant IL-1β production may explain the presence of bone lesions in the patients with IgM MM.

Reduction in C-reactive protein indicates successful targeting of the IL-1/IL-6 axis resulting in improved survival in early stage multiple myeloma.

We report the long‐term follow‐up results of a phase II trial of IL‐1 receptor antagonist and low‐dose dexamethasone for early stage multiple myeloma (MM). Patients were eligible if they had smoldering multiple myeloma (SMM) or indolent multiple myeloma (IMM) without the need for immediate therapy. Forty seven patients were enrolled and subsequently treated with IL‐1Ra; in 25/47 low‐dose dexamethasone (20 mg weekly) was added. The primary endpoint was progression‐free survival (PFS). In the clinical trial, three patients achieved a minor response (MR) to IL‐1Ra alone; five patients a partial response (PR) and four patients an MR after addition of dexamethasone. Seven patients showed a decrease in the plasma cell labeling index (PCLI) which paralleled a decrease in the high sensitivity C‐reactive protein (hs‐CRP). The median PFS for the 47 patients was 1116 days (37.2 months). The median PFS for patients without (n = 22) and with (n = 25) a decrease in their baseline hs‐CRP was 326 days (11 months) vs. 3139 days (104 months) respectively (P <0.0001). The median overall survival (OS) for the 47 patients was 3482 days (9.5 years). The median OS for patients without and with a decrease in their baseline hs‐CRP was 2885 days (7.9 years) vs. median not reached, respectively (P = 0.001). In SMM/IMM patients at risk for progression to active myeloma, reduction in the hs‐CRP indicates successful targeting of the IL‐1/IL‐6 axis resulting in improved PFS and OS.

TRANSCRIPTIONAL CONTROL OF MHC CLASS II ANTIGEN EXPRESSION ON MOUSE T CELL LINES

We have analysed the factors which regulate MHC class II expression in mouse T cell lines. Two such lines, BW 5147 and PLT‐24.2, were used in this study. Using 5‐azacytidine (5 AzaC) we have shown that hypomethylation of DNA can induce class II antigen synthesis in BW 5147. The expression of class II in PLT‐24.2 cells seems to be under a different control mechanism. Southern blot analysis of I‐Aβ gene in PLT‐24.2 suggests that the expression of class II in this cell line is probably the outcome of a gene rearrangement. We hypothesise that insertion of viral long terminal repeats (LTR) next to the class II genes in transformed T cell lines can act as a promoter for the expression of class II antigens.

Mutation in the Aβ gene of B6.C-H-2bm12 generates unique T-cell recognition sites

Four functional Ia genes are coded within the I (immune response) region of mouse, e. g., A~, A~, E~, and E~ (Jones et al. 1978, Klein et al. 1981). In recent years using congenic inbred and recombinant strains of mice, several investigators have demonstrated a relationship between Ia molecules and immune response. The findings on the only known Ia mutant, B6.C-H-2 b.a2, have helped in further characterization of fine specificities on an la molecule. Tryptic peptide maps revealed a limited number of differences between B6 and bm12, mapping the mutation to the A beta polypeptide chain (McKean et al. 1981). The tryptic peptide studies showed that this mutation is not due to a single-point mutation, but involves a minimum of three different amino acid substitutions. Recently, McIntyre and Seidman (1984) have cloned and sequenced a gene coding the bml2 A-beta polypeptide chain. Three nucleotide substitutions result in three amino acid substitutions within a span of five amino acids; isoleucine at position 67, arginine at position 70, and threonine at position 71 are being substituted by phenylalanine, glutamine, and lysine respectively. Two of these amino acid substitutions (arginine-glutamine and threonine-lysine) are nonconservative changes in the tertiary structure of the beta polypeptide chain resulting in the observed alterations in the serology and function of the bml2 A molecule. Amino acid sequence comparison also shows that two of the amino acid substitutions found in the bml2 A;~ chain are found at the same positions in several human class II beta chains (McIntyre and Seidman 1984). These comparisons suggest that the bml2 mutation in the A beta gene arose from a gene-conversion-type event in which another class II beta chain gene acted as a donor sequence. Comparison of the bml2 nucleotide substitutions to the sequence of a cosmid clone for the E~ gene has suggested that the E~ served as this donor sequence (Widera and Flavell 1984), where a minimum of 14 nucleotides of the A;~ gene are

Abstract 2582: P2X7 receptor is functional in myeloma cell lines and myeloma patient cells and can be modulated by P2X7 receptor antagonists: Implications for myeloma therapy

Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1β processing and secretion. We investigated the role this receptor may play in the IL-1/IL-6 cytokine pathway in multiple myeloma. A panel of multiple myeloma cell lines was evaluated by RT-PCR for the expression of P2X7R mRNA. Levels of expression in ANBL-6, MM1S, U266, RPMI-8226 and KAS-6/1 were comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from 1mM to 5mM. Three P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 3 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition. In addition fresh patient samples (15 multiple myelomas/ 4 smoldering myelomas/8 MGUS) were evaluated for inhibition of IL-1β release by the P2X7R antagonists through direct measurement of IL-1β by ELISA as well as by an indirect measurement of IL-1β induced IL-6 production by normal bone marrow stromal cells. Greater than 90% inhibition of IL-1β release was demonstrated in all MM & SMM patients positive for IL-1β expression. IL-1β was not detected in any of the MGUS patients and therefore no affect of the inhibitors was observed. These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2011-2582

Abstract 2449: A novel humanized anti-IL-1beta antibody is highly effective at inhibiting IL-1 induced IL-6 production in myeloma patients in vitro

Background: IL-6 is a central myeloma growth factor and in vitro abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6. Mimicking in vitro observations, we have shown that responsive early stage myeloma patients at risk for progression to active myeloma who were treated with IL-1 inhibitors demonstrated decreases in the myeloma proliferative rate and C-reactive protein (IL-6 surrogate marker) leading to a chronic disease state with an improved progression free survival. In this study we investigated the effect of XOMA 052, a humanized anti-IL-1 antibody to inhibit stromal cell IL-6 production induced by either recombinant IL-1beta or supernatants generated from unsorted bone marrow cells from patients with smoldering multiple myeloma. Methods: Bone marrow stromal cells were incubated with varying concentrations of recombinant IL-1β (100, 10, 1, 0.1 pg/ml) in the absence or presence of anti-IL-1 antibody, anti-TNF antibody, or dexamethasone. In a similar fashion, supernatants from unsorted bone marrow cells from six patients with smoldering myeloma were co-cultured with bone marrow stromal cells and the effect of the humanized anti-IL-1β antibody was tested. Results: The anti-IL-1β antibody inhibited the IL-1 induced IL-6 production in vitro by > 85% at 100 pg/ml of IL-1 and > 90% at 10, 1, and 0.1 pg/ml of IL-1; specifically, using 10 pg/ml of IL-1, IL-6 production was inhibited from 149 down to 5.2 ng/ml in the presence of anti-IL-1 antibody. Anti-TNF antibody had minimal effect. At 10 pg/ml of IL-1 beta, a physiologically relevant amount of IL-1 in vitro in bone marrow samples from myeloma patients, the anti-IL-1 antibody was superior to dexamethasone at inhibition of IL-6 production. In in vitro testing of patient supernatants from unsorted bone marrow cells, three patients were high inducers and three patients low inducers of paracrine IL-6 production. The results demonstrate > 85% reduction of IL-6 production in all patients9 samples tested. Importantly, the anti-IL-1β antibody was highly effective in samples from the three patients who were high inducers of paracrine IL-6 production (IL-6 production was inhibited from 191 down to 12 ng/ml; 157 to 13 ng/ml and 320 to 11 ng/ml). In comparison, the anti-TNF antibody had marginal effects. Conclusion: The humanized anti-IL-1beta antibody is highly effective in vitro at the inhibition of IL-6 generated by supernatant cultures from patients with early stage myeloma or by recombinant IL-1. Combination therapy with IL-1 inhibitors and apoptosis inducing agents may be useful in patients with active myeloma that have elevated IL-6 levels and a high growth rate at diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2449.