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.

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.

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.

Fibroblasts isolated from common sites of breast cancer metastasis enhance cancer cell growth rates and invasiveness in an interleukin-6-dependent manner.

Common sites of breast cancer metastasis include the lung, liver, and bone, and of these secondary metastatic sites, estrogen receptor alpha (ERalpha)-positive breast cancer often favors bone. Within secondary organs, cancer cells would predictably encounter tissue-specific fibroblasts or their soluble factors, yet our understanding of how tissue-specific fibroblasts directly affect cancer cell growth rates and survival remains largely unknown. Therefore, we tested the hypothesis that mesenchymal fibroblasts isolated from common sites of breast cancer metastasis provide a more favorable microenvironment with respect to tumor growth rates. We found a direct correlation between the ability of breast, lung, and bone fibroblasts to enhance ERalpha-positive breast cancer cell growth and the level of soluble interleukin-6 (IL-6) produced by each organ-specific fibroblast, and fibroblast-mediated growth enhancement was inhibited by the removal or inhibition of IL-6. Interestingly, mice coinjected with MCF-7 breast tumor cells and senescent skin fibroblasts, which secrete IL-6, developed tumors, whereas mice coinjected with presenescent skin fibroblasts that produce little to no IL-6 failed to form xenograft tumors. We subsequently determined that IL-6 promoted growth and invasion of breast cancer cells through signal transducer and activator of transcription 3-dependent up-regulation of Notch-3, Jagged-1, and carbonic anhydrase IX. These data suggest that tissue-specific fibroblasts and the factors they produce can promote breast cancer disease progression and may represent attractive targets for development of new therapeutics.

NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma.

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

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.

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.

Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential.

For reasons that are not apparent, it has been difficult to isolate and expand the adult stem cells referred to as mesenchymal stem cells or marrow stromal cells (MSCs) from murine bone marrow. We developed a protocol that provides rapidly expanding MSCs from 5 strains of inbred mice. The MSCs obtained from 5 different strains of mice were similar to human and rat MSCs in that they expanded more rapidly if plated at very low density, formed single-cell-derived colonies, and readily differentiated into either adipocytes, chondrocytes, or mineralizing cells. However, the cells from the 5 strains differed in their media requirements for optimal growth, rates of propagation, and presence of the surface epitopes CD34, stem cell antigen-1 (Sca-1), and vascular cell adhesion molecule 1 (VCAM-1). The protocol should make it possible to undertake a large number of experiments with MSCs in transgenic mice that have previously not been possible. The differences among MSCs from different strains may explain some of the conflicting data recently published on the engraftment of mouse MSCs or other bone marrow cells into nonhematopoietic tissues.

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.