Citizen Science Uncovers Phytophthora ramorum as a Threat to Several Rare or Endangered California Manzanita Species.

The Sudden Oak Death (SOD) Blitzes consist of yearly surveys led by citizen scientists designed to map the distribution of Phytophthora ramorum, cause of the forest disease called SOD, across northern California. During the 2017 Santa Cruz County SOD Blitz, six rare or endangered Arctostaphylos (manzanita) species were found to be possibly symptomatic for the first time. Symptoms included branch cankers and associated canopy mortality, and affected multiple individuals per species. Isolates of P. ramorum were obtained from each of the six species and, through a 30-day-long inoculation experiment on live plants, Koch's postulates were completed for each one of them, conclusively determining that they all are hosts of this pathogen. Two additional manzanita species were later found to be apparently symptomatic in Marin County. Inoculations on detached branches using an isolate of P. ramorum obtained from one of the six rare species from Santa Cruz County were successful, suggesting that these two species may also be hosts of P. ramorum. Detached leaves of all eight species were also successfully inoculated at the University of California-Berkeley in fall 2018 and then again in spring 2019. In these cases, the same isolate was used for all inoculations, in order to obtain information on the comparative susceptibility of the eight species in question. Both branch and leaf inoculations identified significant interspecific differences in susceptibility. The production of sporangia was low on all species but it was not zero, suggesting that sporulation may cause within-plant and limited across-plant contagion, especially in rainy years.

Differences in Hospital Billing for Total Joint Arthroplasty Based on Hospital Profit Status.

BACKGROUND: Regional variations in hospital billing for total joint arthroplasty (TJA) have been reported. It is not clear whether differences exist in hospital charges for TJA based on hospital profit status. METHODS: Data from the Centers for Medicare and Medicaid Services on Medicare Severity-Diagnosis Related Groups (MS-DRGs) 469 (TJA with comorbidity) and 470 (TJA without comorbidity) for fiscal year 2011 were analyzed. Differences in hospital charges and payments were investigated based on hospital profit status (nonprofit, government, and proprietary). Generalized estimating equations determined differences in charges and reimbursement between hospital types controlling for census region, MS-DRG, and number of discharges. RESULTS: Significant differences in billing between institutions existed with median average hospital charges for nonprofit, government, and proprietary institutions being $70,514.30, $73,540.99, and $113,203.77 (P < .0001), respectively, for DRG 469 and $45,363.95, $44,956.57, and $62,715.39 (P < .0001), respectively, for DRG 470. Median average Centers for Medicare and Medicaid Services payments for nonprofit, government, and proprietary institutions for DRG 469 were $22,334.34, $21,346.65, and $21,281.30 (P = .017), respectively, and $14,461.95, $14,466.04, and $13,733.62 (P < .0001), respectively, for DRG 470. Multivariate analyses indicate that nonprofit hospitals charge 5% more (P = .021) and receive 3% less (P = .011) reimbursement than government hospitals. Proprietary hospitals charge 34% more (P < .0001) and receive 7% less (P < .0001) reimbursement than government hospitals. CONCLUSION: Significant differences in hospital charges based on institution profit status were found, with proprietary institutions charging significantly more than nonprofit and government institutions. However, proprietary institutions had the lowest median average reimbursement.

Development and validation of panoptic Meso scale discovery assay to quantify total systemic interleukin-6.

AIM: Interleukin-6 (IL-6), a multifunctional cytokine, exists in several forms ranging from a low molecular weight (MW 20-30 kDa) non-complexed form to high MW (200-450 kDa), complexes. Accurate baseline IL-6 assessment is pivotal to understand clinical responses to IL-6-targeted treatments. Existing assays measure only the low MW, non-complexed IL-6 form. The present work aimed to develop a validated assay to measure accurately total IL-6 (complexed and non-complexed) in serum or plasma as matrix in a high throughput and easily standardized format for clinical testing. METHODS: Commercial capture and detection antibodies were screened against humanized IL-6 and evaluated in an enzyme-linked immunosorbent assay format. The best antibody combinations were screened to identify an antibody pair that gave minimum background and maximum recovery of IL-6 in the presence of 100% serum matrix. A plate-based total IL-6 assay was developed and transferred to the Meso Scale Discovery (MSD) platform for large scale clinical testing. RESULTS: The top-performing antibody pair from 36 capture and four detection candidates was validated on the MSD platform. The lower limit of quantification in human serum samples (n = 6) was 9.77 pg l(-1) , recovery ranged from 93.13-113.27%, the overall pooled coefficients of variation were 20.12% (inter-assay) and 8.67% (intra-assay). High MW forms of IL-6, in size fractionated serum samples from myelodysplastic syndrome and rheumatoid arthritis patients, were detected by the assay but not by a commercial kit. CONCLUSION: This novel panoptic (sees all forms) IL-6 MSD assay that measures both high and low MW forms may have clinical utility.

A phase 2 multicentre study of siltuximab, an anti-interleukin-6 monoclonal antibody, in patients with relapsed or refractory multiple myeloma.

Interleukin-6 (IL6) plays a central role in multiple myeloma pathogenesis and confers resistance to corticosteroid-induced apoptosis. We therefore evaluated the efficacy and safety of siltuximab, an anti-IL6 monoclonal antibody, alone and in combination with dexamethasone, for patients with relapsed or refractory multiple myeloma who had ≥ 2 prior lines of therapy, one of which had to be bortezomib-based. Fourteen initial patients received siltuximab alone, 10 of whom had dexamethasone added for suboptimal response; 39 subsequent patients were treated with concurrent siltuximab and dexamethasone. Patients received a median of four prior lines of therapy, 83% were relapsed and refractory, and 70% refractory to their last dexamethasone-containing regimen. Suppression of serum C-reactive protein levels, a surrogate marker of IL6 inhibition, was demonstrated. There were no responses to siltuximab but combination therapy yielded a partial (17%) + minimal (6%) response rate of 23%, with responses seen in dexamethasone-refractory disease. The median time to progression, progression-free survival and overall survival for combination therapy was 4.4, 3.7 and 20.4 months respectively. Haematological toxicity was common but manageable. Infections occurred in 57% of combination-treated patients, including ≥ grade 3 infections in 18%. Further study of siltuximab in modern corticosteroid-containing myeloma regimens is warranted, with special attention to infection-related toxicity.

Risk factors for oxygen toxicity seizures in hyperbaric oxygen therapy: case reports from multiple institutions.

Oxygen toxicity seizures are a rare but recognized complication of hyperbaric oxygen (HBO2) therapy. Many patients undergoing HBO2 therapy have medical conditions or are taking medications that could contribute to seizures. Previous literature has not extensively reported on these factors in patients experiencing oxygen toxicity seizures. We conducted a chart review at several hyperbaric oxygen centers in the Milwaukee, Wisc., area to explore whether the patients who experienced seizures in the hyperbaric chamber had other medical comorbidities or were on medications which lowered their seizure threshold, thereby contributing to oxygen toxicity seizures. There were a total of seven cases of seizures in five patients. Each patient had risk factors for seizures, including hypercapnia secondary to chronic obstructive pulmonary disease, narcotic withdrawal, alcohol dependence, and antidepressant, tramadol or cephalosporin/ceftriaxone use. We hypothesize that patients who experience oxygen toxicity seizures may have other factors which contribute to the development of these seizures.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

The role of Dickkopf-1 in bone development, homeostasis, and disease.

Wnt/beta-catenin signaling is central to bone development and homeostasis in adulthood and its deregulation is associated with bone pathologies. Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/beta-catenin signaling required for embryonic head development, regulates Wnt signaling by binding to the Wnt coreceptor lipoprotein-related protein-5 (LRP5)/Arrow. LRP5 mutations causing high bone mass syndromes disrupt DKK1-mediated regulation of LRP5. Forced overexpression of Dkk1 in osteoblasts causes osteopenia, disruption of the hematopoietic stem cell (HSC) niche, and defects in HSC function. Dkk1 also inhibits fracture repair. Studies suggest that DKK1 activation in osteoblasts is the underlying cause of glucocorticoid- and estrogen deficiency-mediated osteoporosis, and at least partially underlies the teratogenic effects of thalidomide on limb development. DKK1 induces proliferation of mesenchymal stem cells (MSC) in vitro and may play a role in the development of high-grade undifferentiated pleomorphic sarcomas derived from MSC and osteosarcomas. DKK1 has been implicated in causing erosive arthritis, the osteolytic phenotypes of multiple myeloma and metastatic breast cancer, and osteoblastic metastases of prostate cancer. Preclinical studies have shown that neutralizing DKK1/Dkk1 and/or enhancing Wnt/beta-catenin signaling may prove effective in treating bone pathologies. Here, we review the rapidly growing body of literature defining a pivotal role for DKK1 in bone health and disease.

STAT3 can be activated through paracrine signaling in breast epithelial cells.

BACKGROUND: Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood. METHODS: Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis. RESULTS: Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells. CONCLUSION: These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with cell proliferation. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic regimens for breast carcinomas and other cancers with elevated p-STAT3 levels.

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

Bone is the primary anatomical site of breast cancer metastasis, and bone metastasis is associated with increased morbidity and mortality. Mesenchymal 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 (ERalpha)-positive (MCF-7, T47D, BT474, and ZR-75-1) and ERalpha-negative (MDA-MB-231 and MDA-MB-468) human breast tumor cell lines. All ERalpha-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 phosphorylation 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 ERalpha-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.

Human bone marrow stromal cells enhance breast cancer cell growth rates in a cell line-dependent manner when evaluated in 3D tumor environments.

Our understanding of the impact that fibroblasts have on cancer cell behavior in vivo has been limited by the complexities of in vivo tumor microenvironments, which contain many distinct cell populations that influence tumor growth and survival. Herein, we describe a novel, three-dimensional (3D), in vitro, fluorometric, Tumor Growth Assay (TGA) that allows for non-invasive measurements of cancer cell expansion in the presence of multiple tumor-associated cell types or soluble factors, while embedded in Cultrex or Matrigel Basement Membrane Extract (BME). Using this assay, we investigated the direct biological impact of primary human bone marrow stromal cells (hMSC) on the growth rates of a panel of metastatic breast cancer cell lines. Human MSC can be readily isolated from bone marrow, a principle site of breast cancer metastasis, and were found to significantly enhance the growth rate of MCF-7 (P-value<0.0001), an estrogen receptor-alpha (ERalpha) positive breast cancer cell line, in a soluble factor-dependent manner. MSC paracrine factors also enhanced the growth of other ERalpha positive breast cancer cell lines including T47D, BT474, and ZR-75-1 (P-value<0.05). In contrast, the ERalpha negative cell line MDA-MB-231 was unaffected by hMSC and the growth rate of another ERalpha negative cell line MDA-MB-468 was elevated in the presence of hMSC, albeit to a lesser extent than MCF-7 or the other ERalpha positive cell lines tested.

Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles.

Recent evidence suggests that mesenchymal stem cells (MSC) selectively home to tumors, where they contribute to the formation of tumor-associated stroma. This effect can be opposed by genetically modifying MSC to produce high levels of anti-cancer agents that blunt tumor growth kinetics and inhibit the growth of tumors in situ. In this review article, we describe the biological properties of MSC within the tumor microenvironment and discuss the potential use of MSC and other bone marrow-derived cell populations as delivery vehicles for antitumor proteins.

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.

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.

Analysis of Inflammatory and Anemia-Related Biomarkers in a Randomized, Double-Blind, Placebo-Controlled Study of Siltuximab (Anti-IL6 Monoclonal Antibody) in Patients With Multicentric Castleman Disease.

PURPOSE: Siltuximab (IL6 antibody) is approved for the treatment of multicentric Castleman disease (MCD). Effects of IL6 inhibition on the inflammatory milieu accompanying MCD have not been characterized. EXPERIMENTAL DESIGN: Trends in inflammatory- and anemia-associated markers, measured over the course of a placebo-controlled study of siltuximab (11 mg/kg q3w) in patients with MCD (n = 79), were characterized. RESULTS: Baseline IL6 and C-reactive protein (CRP) levels were significantly correlated (r = 0.708; P < 0.0001). CRP levels decreased (median, 92%) by cycle 1 day 8 (C1D8), remaining suppressed during siltuximab treatment while remaining stable in the placebo group. There were no associations between baseline CRP or IL6 and MCD symptom burden, histologic subtype, ethnicity, maximum CRP decrease, and response parameters. A hemoglobin response (change ≥ 15 g/L at week 13) was observed with siltuximab (61%; P = 0.0002). Median hepcidin decrease from baseline at C1D8 with siltuximab was 47% versus median 11% increase with placebo. Maximum post-baseline changes in hepcidin levels among siltuximab recipients were correlated with maximum changes for hemoglobin (r = -0.395; P = 0.00607), total iron-binding capacity (TIBC; r = -0.354; P = 0.01694), and ferritin (r = 0.599; P = 0.0001). Greater median changes from baseline in ferritin, hemoglobin, and TIBC were observed in anemic siltuximab-treated patients. CONCLUSIONS: IL6 neutralization with siltuximab resulted in sustained CRP suppression and improvement of anemia, in part, by hepcidin pathway inhibition.

Stromal cells expressing elevated VCAM-1 enhance survival of B lineage tumor cells.

In the current study we generated murine bone marrow stromal cells that constitutively express human VCAM-1. These stromal cell lines allow investigation of the contribution of VCAM-1 initiated signaling to tumor cell survival. Co-culture of ALL cell lines with stromal cells engineered to over express VCAM-1 enhanced survival of leukemic cells in a PI-3 kinase-dependent manner, compared to co-culture with parental stromal cells expressing only endogenous VCAM-1. These observations suggest that modulation of stromal cell VCAM-1 by specific chemotherapeutic drugs may have utility in decreasing residual disease. In addition, these novel lines provide an in vitro model in which other tumor types that interact with stromal cells in the bone marrow microenvironment may be evaluated to determine the contribution of VCAM-1 initiated signaling to modulation of treatment response.

Regulation of lymphoid and myeloid leukemic cell survival: role of stromal cell adhesion molecules.

Several laboratories have documented the necessity for direct contact of lymphoid and myeloid leukemic cells with bone marrow stromal cells for optimal survival. Subsequent studies have identified various stromal cell adhesion molecules and soluble factors that facilitate survival through leukemic cell anti-apoptotic signal transduction pathways. This report provides an overview of enhanced leukemic cell survival through adhesive interactions with bone marrow expressed molecules. In addition, we describe the establishment of cloned murine stromal cell lines engineered to constitutively express human VCAM-1 protein on their surface. These stromal cell lines will be useful in studies aimed at better understanding the specific contribution of VCAM-1: VLA-4 signaling in maintenance of residual leukemic disease.

Chemotherapy induces bcl-2 cleavage in lymphoid leukemic cell lines.

Bcl-2 is the major anti-apoptotic protein evaluated in studies aimed at understanding programmed cell death. Recent work suggests that the biological activity of Bcl-2 is modulated by proteolytic cleavage, with a 23 kDa cleaved Bcl-2 product having pro-apoptotic activity. In the current study we evaluated the effect of chemotherapy on Bcl-2 cleavage in B lineage leukemic cell lines. JM-1, SUP-B 15 and RS4 leukemic cell lines cleaved Bcl-2 to its 23 kDa form when exposed to the chemotherapeutic agents 1-beta-D-arabinofuranosyl-cytosine (Ara-C) or etoposide (VP-16). Chemotherapy induced Bcl-2 cleavage was blunted by inhibition of caspase activity. Co-culture of leukemic cells with bone marrow stromal cells during chemotherapy exposure resulted in reduced levels of 23 kDa Bcl-2 protein. These observations suggest that the bone marrow microenvironment may contribute to maintenance of residual leukemic disease during treatment by reducing generation of pro-apoptotic 23 kDa Bcl-2.

A phase I/II, multiple-dose, dose-escalation study of siltuximab, an anti-interleukin-6 monoclonal antibody, in patients with advanced solid tumors.

PURPOSE: This phase I/II study evaluated safety, efficacy, and pharmacokinetics of escalating, multiple doses of siltuximab, a chimeric anti-interleukin (IL)-6 monoclonal antibody derived from a new Chinese hamster ovary (CHO) cell line in patients with advanced/refractory solid tumors. EXPERIMENTAL DESIGN: In the phase I dose-escalation cohorts, 20 patients with advanced/refractory solid tumors received siltuximab 2.8 or 5.5 mg/kg every 2 weeks or 11 or 15 mg/kg every 3 weeks intravenously (i.v.). In the phase I expansion (n = 24) and phase II cohorts (n = 40), patients with Kirsten rat sarcoma-2 (KRAS)-mutant tumors, ovarian, pancreatic, or anti-EGF receptor (EGFR) refractory/resistant non-small cell lung cancer (NSCLC), colorectal, or H&N cancer received 15 mg/kg every 3 weeks. The phase II primary efficacy endpoint was complete response, partial response, or stable disease >6 weeks. RESULTS: Eighty-four patients (35 colorectal, 29 ovarian, 9 pancreatic, and 11 other) received a median of three (range, 1-45) cycles. One dose-limiting toxicity occurred at 5.5 mg/kg. Common grade ≥3 adverse events were hepatic function abnormalities (15%), physical health deterioration (12%), and fatigue (11%). Ten percent of patients had siltuximab-related grade ≥3 adverse events. Neutropenia (4%) was the only possibly related adverse event grade ≥3 reported in >1 patient. Serious adverse events were reported in 42%; most were related to underlying disease. The pharmacokinetic profile of CHO-derived siltuximab appears similar to the previous cell line. No objective responses occurred; 5 of 84 patients had stable disease >6 weeks. Hemoglobin increased ≥1.5 g/dL in 33 of 47 patients. At 11 and 15 mg/kg, completely sustained C-reactive protein suppression was observed. CONCLUSIONS: Siltuximab monotherapy appears to be well tolerated but without clinical activity in solid tumors, including ovarian and KRAS-mutant cancers. The recommended phase II doses were 11 and 15 mg/kg every 3 weeks.

Interleukin-6 receptor polymorphism is prevalent in HIV-negative Castleman Disease and is associated with increased soluble interleukin-6 receptor levels.

Multicentric Castleman Disease is largely driven by increased signaling in the pathway for the plasma cell growth factor interleukin-6. We hypothesized that interleukin-6/interleukin-6 receptor/gp130 polymorphisms contribute to increased interleukin-6 and/or other components of the interleukin-6 signaling pathway in HIV-negative Castleman Disease patients. The study group was composed of 58 patients and 50 healthy donors of a similar racial/ethnic profile. Of seven polymorphisms chosen for analysis, we observed an increased frequency between patients and controls of the minor allele of interleukin-6 receptor polymorphism rs4537545, which is in linkage disequilibrium with interleukin-6 receptor polymorphism rs2228145. Further, individuals possessing at least one copy of the minor allele of either polymorphism expressed higher levels of soluble interleukin-6 receptor. These elevated interleukin-6 receptor levels may contribute to increased interleukin-6 activity through the trans-signaling pathway. These data suggest that interleukin-6 receptor polymorphism may be a contributing factor in Castleman Disease, and further research is warranted.

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.