Amy Axel

Interleukin-6 is a potent growth factor for ER-α-positive human breast cancer

Bone is the primary anatomical site of breast cancer metastasis, and bone metastasis is associated with increased morbidity and mortality. Mesenchy‐mal stem cells (MSC) are a predominant fibroblast cell population within the bone marrow, and metastatic breast cancer cells that seed within bone would predictably encounter MSC or their soluble factors. Therefore, we examined the impact of primary human MSC on a panel of estrogen receptor‐alpha (ERα)‐positive (MCF‐7, T47D, BT474, and ZR‐75–1) and ERα‐negative (MDA‐MB‐231 and MDA‐MB‐468) human breast tumor cell lines. All ERα‐positive breast tumor cell lines displayed low basal activation of signal transducer and activator of transcription 3 (STAT3) until exposed to MSC, which induced chronic phosphorylation of STAT3 on tyrosine‐705. Paracrine IL‐6 was found to be the principal mediator of STAT3 phosphorylation in coculture studies, and MSC induction of STAT3 phos‐phorylation was lost when IL‐6 was depleted from MSC conditioned media or the IL‐6 receptor was blocked on tumor cells. Enhanced tumor cell growth rates were observed in the ERα‐positive mammary tumor cell line MCF‐7 after paracrine and autocrine IL‐6 exposure, where MCF‐7 growth rates were enhanced by > 2‐fold when cocultured with MSC in vitro and even more pronounced in vivo with autocrine IL‐6 production.—Sasser, A. K., Sullivan, N. J., Studebaker, A. W., Hendey, L. F., Axel, A. E., Hall, B. M. Interleukin‐6 is a potent growth factor for ER‐α‐positive human breast cancer. FASEB J. 21, 3763–3770 (2007)

Clinical efficacy of daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma

The efficacy and favorable safety profile of daratumumab monotherapy in multiple myeloma (MM) was previously reported. Here, we present an updated pooled analysis of 148 patients treated with daratumumab 16 mg/kg. Data were combined from part 2 of a first-in-human phase 1/2 study of patients who relapsed after or were refractory to ≥2 prior therapies and a phase 2 study of patients previously treated with ≥3 prior lines of therapy (including a proteasome inhibitor [PI] and an immunomodulatory drug [IMiD]) or were double refractory. Among the pooled population, patients received a median of 5 prior lines of therapy (range, 2 to 14 prior lines of therapy), and 86.5% were double refractory to a PI and an IMiD. Overall response rate was 31.1%, including 13 very good partial responses, 4 complete responses, and 3 stringent complete responses. The median duration of response was 7.6 months (95% confidence interval [CI], 5.6 to not evaluable [NE]). The median progression-free survival (PFS) and overall survival (OS) were 4.0 months (95% CI, 2.8-5.6 months) and 20.1 months (95% CI, 16.6 months to NE), respectively. When stratified by responders vs stable disease/minimal response vs progressive disease/NE, median PFS was 15.0 months (95% CI, 7.4 months to NE) vs 3.0 months (95% CI, 2.8-3.7 months) vs 0.9 months (95% CI, 0.9-1.0 months), respectively, and median OS was NE (95% CI, NE to NE) vs 18.5 months (95% CI, 15.1-22.4 months) vs 3.7 months (95% CI, 1.7-7.6 months), respectively. No new safety signals were identified. In this pooled data set, daratumumab 16 mg/kg monotherapy demonstrated rapid, deep, and durable responses, with a clinical benefit that extended to patients with stable disease or better.

CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma

The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow-localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

Monitoring multiple myeloma patients treated with daratumumab: teasing out monoclonal antibody interference

Abstract Background: Monoclonal antibodies are promising anti-myeloma treatments. As immunoglobulins, monoclonal antibodies have the potential to be identified by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). Therapeutic antibody interference with standard clinical SPE and IFE can confound the use of these tests for response assessment in clinical trials and disease monitoring. Methods: To discriminate between endogenous myeloma protein and daratumumab, a daratumumab-specific immunofixation electrophoresis reflex assay (DIRA) was developed using a mouse anti-daratumumab antibody. To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, it was spiked into daratumumab-containing serum and resolved by IFE/SPE. The presence (DIRA positive) or absence (DIRA negative) of residual M-protein in daratumumab-treated patient samples was evaluated using predetermined assessment criteria. DIRA was evaluated for specificity, limit of sensitivity, and reproducibility. Results: In all of the tested samples, DIRA distinguished between daratumumab and residual M-protein in commercial serum samples spiked with daratumumab and in daratumumab-treated patient samples. The DIRA limit of sensitivity was 0.2 g/L daratumumab, using spiking experiments. Results from DIRA were reproducible over multiple days, operators, and assays. The anti-daratumumab antibody was highly specific for daratumumab and did not shift endogenous M-protein. Conclusions: As the treatment of myeloma evolves to incorporate novel monoclonal antibodies, additional solutions will be needed for clinical monitoring of patient responses to therapeutic regimens. In the interim, assays such as DIRA can inform clinical outcomes by distinguishing daratumumab from endogenous M-protein by IFE.

Translation of a Tumor Microenvironment Mimicking 3D Tumor Growth Co-culture Assay Platform to High-Content Screening

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.

Prognostic significance of interleukin-6 single nucleotide polymorphism genotypes in neuroblastoma: rs1800795 (promoter) and rs8192284 (receptor).

Purpose: Neuroblastoma is a childhood cancer of the sympathetic nervous system and many patients present with high-risk disease. Risk stratification, based on pathology and tumor-derived biomarkers, has improved prediction of clinical outcomes, but overall survival (OS) rates remain unfavorable and new therapeutic targets are needed. Some studies suggest a link between interleukin (IL)-6 and more aggressive behavior in neuroblastoma tumor cells. Therefore, we examined the impact of two IL-6 single nucleotide polymorphisms (SNP) on neuroblastoma disease progression. Experimental Design: DNA samples from 96 high-risk neuroblastoma patients were screened for two SNP that are known to regulate the serum levels of IL-6 and the soluble IL-6 receptor, rs1800795 and rs8192284, respectively. The genotype for each SNP was determined in a blinded fashion and independent statistical analysis was done to determine SNP-related event-free survival (EFS) and OS rates. Results: The rs1800795 IL-6 promoter SNP is an independent prognostic factor for EFS and OS in high-risk neuroblastoma patients. In contrast, the rs8192284 IL-6 receptor SNP revealed no prognostic value. Conclusions: The rs1800795 SNP [−174 IL-6 (G > C)] represents a novel and independent prognostic marker for both EFS and OS in high-risk neuroblastoma. Because the rs1800795 SNP [−174 IL-6 (G > C)] has been shown to correlate with production of IL-6, this cytokine may represent a target for development of new therapies in neuroblastoma. (Clin Cancer Res 2009;15(16):5234–9)

Interference of daratumumab in monitoring multiple myeloma patients using serum immunofixation electrophoresis can be abrogated using the daratumumab IFE reflex assay (DIRA)

Abstract Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody (mAb) currently being evaluated in several Phase 2 and 3 clinical studies for the treatment of multiple myeloma (MM). In this clinical case study we demonstrate that daratumumab can be detected as an individual monoclonal band in serum immunofixation electrophoresis (IFE). M-protein follow-up by IFE is part of the International Myeloma Working Group (IMWG) criteria to assess treatment response. Therefore, it is crucial that the daratumumab band is not confused with the endogenous M-protein of the patient during IFE interpretation. Moreover, a significant number of IgG-κ M-proteins co-migrate with daratumumab. Co-migration introduces a bias in the M-protein quantification since pharmacokinetic studies show that daratumumab peak plasma concentrations reach up to 1 g/L. More importantly, co-migration can mask clearance of the M-protein by IFE which is necessary for classification of complete response by IMWG criteria (negative serum IFE). For optimal M-protein monitoring the laboratory specialist needs to be informed when patients receive daratumumab, and it is essential that the laboratory specialist is aware that a slow migrating band in the γ-region in those patients may be derived from the daratumumab. A daratumumab specific IFE reflex assay (DIRA) has been developed and can be utilized to abrogate interference. The here described mAb interference is not limited to daratumumab, and as therapeutic antibodies gain approval and enter into common clinical practice, laboratory specialists will need additional processes to characterize IFE interference and distinguish endogenous M-protein from therapeutic antibodies.

Abstract 2563: Development of clinical assay to mitigate daratumumab, an IgG1κ monoclonal antibody, interference with serum immunofixation (IFE) and clinical assessment of M-protein response in multiple myeloma

Daratumumab is a fully human anti-CD38 IgG1κ monoclonal antibody currently in clinical development for the treatment of multiple myeloma (MM) (PMID:21187443). Novel agents like daratumumab can potentially result in partial or complete clearance of the M-protein or monoclonal immunoglobulin produced by multiple myeloma cells. Monoclonal paraprotein clearance by serum electrophoresis and immunofixation (IFE) is part of the International Multiple Myeloma Working Group (IMWG) criteria for complete response and followed routinely in clinical practice to assess treatment response. The current limit of detection of many serum IFE assays is approximately 150 μg/ml M-protein, which is below the serum concentration of most monoclonal antibodies dosed in the therapeutic range. Indeed, recent studies demonstrated that therapeutic monoclonal antibodies in clinical development for treatment of MM are readily detected on serum IFE and can interfere with the detection and monitoring of endogenous M-protein (PMID: 20940329, 21521182). Therefore, mitigation strategies will need to be put in place to remove this interference to ensure valid clinical response calls that meet IMWG criteria for complete response and stringent complete response (negative serum IFE). We first confirmed daratumumab9s interference on serum IFE by spiking various concentrations of antibody into PBS and normal healthy serum. We were able to detect daratumumab in PBS and serum at 50 μg/ml. We then utilized commercially-sourced MM serum samples to determine the M-protein profiles that may be observed in patients undergoing treatment with daratumumab and project the percentage of subjects that may demonstrate interference by serum IFE. Daratumumab was spiked into MM sera at clinically relevant doses (200 μg/ml) and the interference was noted in approximately 50% of IgG kappa samples where daratumumab and endogenous M-protein co-migrated. In remaining IgG kappa samples, the endogenous M-protein migrated in a different position than daratumumab and was easily identifiable. To mitigate interference, we utilized a mouse anti-daratumumab antibody to bind daratumumab and shift the complex migration on IFE. In addition, labeling the anti-idiotype antibody with either biotin or Alexa-fluor tags provided further shift of the daratumumab-complex allowing for additional distinction between the therapeutic antibody and endogenous M-protein. Patients with suspected daratumumab interference can be monitored with this additional reflex serum IFE utilizing the mitigation strategy described above. After clinically validating this assay, we intend to incorporate this mitigation strategy in clinical trials with daratumumab to ensure accurate assessment of clinical M-protein response, a critical evaluation for clinical trial endpoints and disease assessment. Citation Format: Amy E. Axel, Christopher R. McCudden, Hong Xie, Brett M. Hall, A. Kate Sasser. Development of clinical assay to mitigate daratumumab, an IgG1κ monoclonal antibody, interference with serum immunofixation (IFE) and clinical assessment of M-protein response in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2014-2563

Effects of daratumumab on natural killer cells and impact on clinical outcomes in relapsed or refractory multiple myeloma

Daratumumab, a human CD38 imunoglobulin G 1κ monoclonal antibody, has demonstrated clinical activity and a manageable safety profile in monotherapy and combination therapy clinical trials in relapsed and/or refractory multiple myeloma. CD38 is expressed at high levels on myeloma cells and, to a lesser extent, on immune effector cells, including natural killer (NK) cells, which are important for daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Here, the pharmacodynamic effects of daratumumab monotherapy on NK cells, and the effect of NK cell dynamics on daratumumab efficacy and safety, were assessed. Daratumumab, like other CD38 antibodies, reduced NK-cell counts in peripheral blood mononuclear cells (PBMCs) of healthy donors in vitro. Data on NK-cell counts, clinical efficacy, and adverse events were pooled from two single-agent daratumumab studies, GEN501 and SIRIUS. In daratumumab-treated myeloma patients, total and activated NK-cell counts reduced rapidly in peripheral blood after the first dose, remained low over the course of treatment, and recovered after treatment ended. There was a clear maximum effect relationship between daratumumab dose and maximum reduction in NK cells. Similar reductions were observed in bone marrow. PBMCs from daratumumab-treated patients induced lysis by ADCC of CD38+ tumor cells in vitro, suggesting that the remaining NK cells retained cytotoxic functionality. There was no relationship between NK-cell count reduction and the efficacy or safety profile of daratumumab. Furthermore, although NK cell numbers are reduced after daratumumab treatment, they are not completely depleted and may still contribute to ADCC, clinical efficacy, and infection control.

Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, estrogen receptor-α-positive breast cancer.

Introduction Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis. Methods The role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERα-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo. Results In eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen. Conclusion Given the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.