Matthew R. Steensma

Atypical subtrochanteric femoral fractures in patients with skeletal malignant involvement treated with intravenous bisphosphonates.

BACKGROUND Atypical subtrochanteric femoral fractures have been identified as a potential complication of long-term bisphosphonate therapy for the treatment of osteoporosis. Patients with skeletal malignant involvement, who receive much higher cumulative doses of bisphosphonates than do patients with osteoporosis, may be at higher risk for the development of these fractures. METHODS A retrospective review of the imaging studies and case notes was done for patients with skeletal malignant involvement who received a minimum of twenty-four doses of intravenous bisphosphonates between 2004 and 2007 and were followed until death or the time of the latest review. Patients were classified as having an atypical subtrochanteric femoral fracture if they had a transverse subtrochanteric fracture following low-energy trauma or an impending fracture, together with radiographic findings of diffuse diaphyseal cortical thickening and cortical beaking at the subtrochanteric area. RESULTS In the study cohort of 327 patients, we identified four patients who developed an atypical subtrochanteric femoral fracture. All four patients were female, three had breast cancer, and one had myeloma. There was no significant difference between patients who developed an atypical subtrochanteric femoral fracture and those who did not with regard to the doses of intravenous bisphosphonates or the duration of treatment. CONCLUSIONS The prevalence of atypical subtrochanteric femoral fractures in patients with skeletal malignant involvement who are managed with high doses of intravenous bisphosphonates is low. All patients in our study who had development of these fractures had prodromal symptoms of thigh pain.

Targeting receptor-activator of nuclear kappaB ligand in aneurysmal bone cysts: verification of target and therapeutic response.

Aneurysmal bone cyst (ABC) is a benign tumor of bone presenting as a cystic, expansile lesion in both the axial and appendicular skeleton. Axial lesions demand special consideration, because treatment-related morbidity can be devastating. In similar lesions, such as giant cell tumor of bone (GCTB), the receptor-activator of nuclear kappaB ligand (RANKL)-receptor-activator of nuclear kappaB (RANK) signaling axis is essential to tumor progression. Although ABC and GCTB are distinct entities, they both contain abundant multinucleated giant cells and are osteolytic characteristically. We hypothesize that ABCs express both RANKL and RANK similarly in a cell-type specific manner, and that targeted RANKL therapy will mitigate ABC tumor progression. Cellular expression of RANKL and RANK was determined in freshly harvested ABC samples using laser confocal microscopy. A consistent cell-type-specific pattern was observed: fibroblastlike stromal cells expressed RANKL strongly whereas monocyte/macrophage precursor and multinucleated giant cells expressed RANK. Relative RANKL expression was determined by quantitative real-time polymerase chain reaction in ABC and GCTB tissue samples; no difference in relative expression was observed (P > 0.05). In addition, we review the case of a 5-year-old boy with a large, aggressive sacral ABC. After 3 months of targeted RANKL inhibition with denosumab, magnetic resonance imaging demonstrated tumor shrinkage, bone reconstitution, and healing of a pathologic fracture. Ambulation, and bowel and bladder function were restored at 6 months. Denosumab treatment was well tolerated. Post hoc analysis demonstrated strong RANKL expression in the pretreatment tumor sample. These findings demonstrate that RANKL-RANK signal activation is essential to ABC tumor progression. RANKL-targeted therapy may be an effective alternative to surgery in select ABC presentations.

Targeting the Giant Cell Tumor Stromal Cell: Functional Characterization and a Novel Therapeutic Strategy

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.

Partial Hand Preservation for Large Soft Tissue Sarcomas of the Hand

PURPOSE Hand amputations cause marked functional loss for patients. In patients with large soft tissue sarcomas of the hand, partial hand preservation is extremely challenging for surgeons attempting a complete resection of the tumor with negative resection margins. We conducted this review to examine the oncologic outcome, including local recurrence rate and patient overall survival, and functional outcome after resections for large soft tissue sarcomas with partial hand preservation. METHODS We performed a retrospective review of all patients with soft tissue sarcomas of the hand treated at our institution from 1995 to 2007. We identified 8 patients who had tumors at least 5 cm in maximum dimension and had tumor resection with partial hand preservation. The mean age at the time of surgery was 49 years (range, 10-80 years). Two patients had myxofibrosarcoma, 2 patients had synovial sarcoma, 2 patients had malignant fibrous histiocytoma, 1 patient had a malignant peripheral nerve sheath tumor, and 1 patient had a liposarcoma. Two patients had low-grade tumors, and 6 patients had high-grade tumors. Two patients had American Joint Committee on Cancer stage 1b tumors, and 6 patients had American Joint Committee on Cancer stage 3 tumors. No patients had distant metastases at the time of surgery. Hand function was evaluated using Musculoskeletal Tumor Society criteria. RESULTS Of the 8 patients, 1 died of distant metastatic disease, 1 developed local tumor recurrence and is alive with locally recurrent disease, and the other 6 patients are completely disease-free. The mean Musculoskeletal Tumor Society score was 26 (range, 19-29), with the 2 patients who had received double-ray amputations having the lower scores (19 and 24). CONCLUSIONS Partial hand preservation is possible in selected patients with large soft tissue sarcomas of the hand, obtaining low local recurrence rates, good overall survival, and good functional outcome. However, all effort should be made to achieve negative resection margins. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic IV.

Down regulation of Wnt signaling mitigates hypoxia-induced chemoresistance in human osteosarcoma cells.

Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6–13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

Skeletal metastasis: treatments, mouse models, and the Wnt signaling.

Skeletal metastases result in significant morbidity and mortality. This is particularly true of cancers with a strong predilection for the bone, such as breast, prostate, and lung cancers. There is currently no reliable cure for skeletal metastasis, and palliative therapy options are limited. The Wnt signaling pathway has been found to play an integral role in the process of skeletal metastasis and may be an important clinical target. Several experimental models of skeletal metastasis have been used to find new biomarkers and test new treatments. In this review, we discuss pathologic process of bone metastasis, the roles of the Wnt signaling, and the available experimental models and treatments.

Oculoectodermal syndrome is a mosaic RASopathy associated with KRAS alterations.

Oculoectodermal syndrome (OES) is a rare disease characterized by a combination of congenital scalp lesions and ocular dermoids, with additional manifestations including non‐ossifying fibromas and giant cell granulomas of the jaw occurring during the first decade of life. To identify the genetic etiology of OES, we conducted whole‐genome sequencing of several tissues in an affected individual. Comparison of DNA from a non‐ossifying fibroma to blood‐derived DNA allowed identification of a somatic missense alteration in KRAS NM_033360.3(KRAS):c.38G>A, resulting in p.Gly13Asp. This alteration was also observed in the patients other affected tissues including the skin and muscle. Targeted sequencing in a second, unrelated OES patient identified an NM_033360.3(KRAS):c.57G>C, p.Leu19Phe alteration. Allelic frequencies fell below 40% in all tissues examined in both patients, suggesting that OES is a mosaic RAS‐related disorder, or RASopathy. The characteristic findings in OES, including scalp lesions, ocular dermoids, and benign tumors, are found in other mosaic and germline RASopathies. This discovery also broadens our understanding of the spectrum of phenotypes resulting from KRAS alterations. Future research into disease progression with regard to malignancy risk and investigation of RAS‐targeted therapies in OES is warranted. KRAS sequencing is clinically available and may also now improve OES diagnostic criteria.

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers.

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients.

Molecular-guided therapy predictions reveal drug resistance phenotypes and treatment alternatives in malignant peripheral nerve sheath tumors

BackgroundMalignant peripheral nerve sheath tumors (MPNST) are rare highly aggressive sarcomas that affect 8-13% of people with neurofibromatosis type 1. The prognosis for patients with MPNST is very poor. Despite TOP2A overexpression in these tumors, doxorubicin resistance is common, and the mechanisms of chemotherapy resistance in MPNST are poorly understood. Molecular-guided therapy prediction is an emerging strategy for treatment refractory sarcomas that involves identification of therapy response and resistance mechanisms in individual tumors. Here, we report the results from a personalized, molecular-guided therapy analysis of MPNST samples.MethodsEstablished molecular-guided therapy prediction software algorithms were used to analyze published microarray data from human MPNST samples and cell lines, with benign neurofibroma tissue controls. MPNST and benign neurofibroma-derived cell lines were used for confirmatory in vitro experimentation using quantitative real-time PCR and growth inhibition assays. Microarray data was analyzed using Affymetrix expression console MAS 5.0 method. Significance was calculated with Welch’s t-test with non-corrected p-value < 0.05 and validated using permutation testing across samples. Paired Student’s t-tests were used to compare relative EC50 values from independent growth inhibition experiments.ResultsMolecular guided therapy predictions highlight substantial variability amongst human MPNST samples in expression of drug target and drug resistance pathways, as well as some similarities amongst samples, including common up-regulation of DNA repair mechanisms. In a subset of MPNSTs, high expression of ABCC1 is observed, serving as a predicted contra-indication for doxorubicin and related therapeutics in these patients. These microarray-based results are confirmed with quantitative, real-time PCR and immunofluorescence. The functional effect of drug efflux in MPNST-derived cells is confirmed using in vitro growth inhibition assays. Alternative therapeutics supported by the molecular-guided therapy predictions are reported and tested in MPNST-derived cells.ConclusionsThese results confirm the substantial molecular heterogeneity of MPNSTs and validate molecular-guided therapy predictions in vitro. The observed molecular heterogeneity in MPNSTs influences therapy prediction. Also, mechanisms involving drug transport and DNA damage repair are primary mediators of MPNST chemotherapy resistance. Together, these findings support the utility of individualized therapy in MPNST as in other sarcomas, and provide initial proof-of concept that individualized therapy prediction can be accomplished.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.