Death by HDAC Inhibition in Synovial Sarcoma Cells.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting the causative SS18-SSX fusion oncoprotein are currently available. Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell-cycle arrest, neuronal differentiation, and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb group targets were reactivated, including tumor suppressor CDKN2A, and proapoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and proapoptotic factors BIK, BIM, and BMF were important to apoptosis induction following HDAC inhibition in synovial sarcoma. HDAC inhibitor pathway activation results in apoptosis and decreased tumor burden following a 7-day quisinostat treatment in the Ptenfl/fl;hSS2 mouse model of synovial sarcoma. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of clinical treatment. Mol Cancer Ther; 16(12); 2656-67. ©2017 AACR.

HDAC and Proteasome Inhibitors Synergize to Activate Pro-Apoptotic Factors in Synovial Sarcoma.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of EGR1 and CDKN2A tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition.

Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D.

BACKGROUND: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. METHODS: Nuclear DNA was stained in 14 microm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of x40 or x100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. RESULTS: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at x60 or x100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. CONCLUSIONS: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

New Strategies in Sarcoma: Linking Genomic and Immunotherapy Approaches to Molecular Subtype.

There are more than 100 sarcoma subtypes, each uncommon and diagnostically challenging. Conventional chemotherapy has little benefit for most soft-tissue sarcomas; new treatment strategies are needed. Multiple recent genomic studies have provided detailed insights into sarcoma biology, including more accurate classification by molecular subtype, identification of recurrent mutations in oncogenic pathways, and evidence of epigenetic dysregulation. Advances in immunotherapy (adoptive immune cell transfer, tumor vaccine strategies, and immune checkpoint inhibition) have also provided a better understanding of how immuno-oncology might best be applied to sarcoma treatment, including connections to oncogenic pathways that may support combination strategies with conventional and targeted therapies. In this article, we review the latest sarcoma genomic studies and immuno-oncology developments and discuss how the findings suggest potential strategies to improve diagnosis and treatment across multiple sarcoma subtypes.

Synovial sarcoma: recent discoveries as a roadmap to new avenues for therapy.

UNLABELLED: Oncogenesis in synovial sarcoma is driven by the chromosomal translocation t(X,18; p11,q11), which generates an in-frame fusion of the SWI/SNF subunit SS18 to the C-terminal repression domains of SSX1 or SSX2. Proteomic studies have identified an integral role of SS18-SSX in the SWI/SNF complex, and provide new evidence for mistargeting of polycomb repression in synovial sarcoma. Two recent in vivo studies are highlighted, providing additional support for the importance of WNT signaling in synovial sarcoma: One used a conditional mouse model in which knockout of ß-catenin prevents tumor formation, and the other used a small-molecule inhibitor of ß-catenin in xenograft models. SIGNIFICANCE: Synovial sarcoma appears to arise from still poorly characterized immature mesenchymal progenitor cells through the action of its primary oncogenic driver, the SS18-SSX fusion gene, which encodes a multifaceted disruptor of epigenetic control. The effects of SS18-SSX on polycomb-mediated gene repression and SWI/SNF chromatin remodeling have recently come into focus and may offer new insights into the basic function of these processes. A central role for deregulation of WNT-ß-catenin signaling in synovial sarcoma has also been strengthened by recent in vivo studies. These new insights into the the biology of synovial sarcoma are guiding novel preclinical and clinical studies in this aggressive cancer.

Confocal 3D DNA cytometry: assessment of required coefficient of variation by computer simulation.

BACKGROUND: Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. So far, sample size has been limited by the time consuming nature of the technology. Since the power of DNA histograms to resolve different stemlines depends on both the sample size and the coefficient of variation (CV) of histogram peaks, interpretation of 3D CLSM DNA histograms might be hampered by both a small sample size and a large CV. The aim of this study was to analyze the required CV for 3D CLSM DNA histograms given a realistic sample size. METHODS: By computer simulation, virtual histograms were composed for sample sizes of 20000, 10000, 5000, 1000, and 273 cells and CVs of 30, 25, 20, 15, 10 and 5%. By visual inspection, the histogram quality with respect to resolution of G0/1 and G2/M peaks of a diploid stemline was assessed. RESULTS: As expected, the interpretability of DNA histograms deteriorated with decreasing sample sizes and higher CVs. For CVs of 15% and lower, a clearly bimodal peak pattern with well distinguishable G0/1 and G2/M peaks were still seen at a sample size of 273 cells, which is our current average sample size with 3D CLSM DNA cytometry. CONCLUSIONS: For unambiguous interpretation of DNA histograms obtained using 3D CLSM, a CV of at most 15% is tolerable at currently achievable sample sizes. To resolve smaller near diploid stemlines, a CV of 10% or better should be aimed at. With currently available 3D imaging technology, this CV is achievable.

Synergism of heat shock protein 90 and histone deacetylase inhibitors in synovial sarcoma.

Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of 17-AAG with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only induce cell death but also activate the survival pathway NF-kappaB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-kappaB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-kappaB activation. In our assays, adding 17-AAG blocks NF-kappaB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-kappaB inhibitor BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-kappaB synergize with HDAC inhibitors against synovial sarcoma.

Kappa statistics to measure interrater and intrarater agreement for 1790 cervical biopsy specimens among twelve pathologists: qualitative histopathologic analysis and methodologic issues.

BACKGROUND: As part of a program project to evaluate emerging optical technologies for cervical neoplasia, we performed fluorescence and reflectance spectroscopic examinations of patients with abnormal Papanicolaou smears. Biopsy specimens were taken from each area and measured optically, and study pathologists performed qualitative histopathologic readings. Several methodologic issues arose in this analysis: (1) the interpathologist and intrapathologist agreement between institutions for the 1790 biopsy specimens; (2) the interinstitutional agreement among the two institutions conducting the trials on 117 randomly chosen biopsy specimens; (3) the interinstitutional agreement among the two institutions and a third expert gynecologic pathologist to ensure the expert readings were comparable to those outside both institutions on 117 randomly chosen biopsy specimens; and (4) an additional three reviews of the 106 difficult biopsy specimens by all three institutions. METHODS: All 1790 specimens from 850 patients were reviewed three times at each institution in blinded fashion; those for which the first and second reviews were identical were not reviewed a third time. A randomly selected sample of 117 specimens was randomly ordered and read by study pathologists at The University of Texas M. D. Anderson Cancer Center, British Columbia Cancer Agency (BCCA), and Brigham and Women's Hospital (BWH). The 106 difficult cases were treated in the same manner as the randomized and random-ordered cases. Generalized, unweighted, and weighted kappas and their 95% confidence intervals were used to assess agreement. Binary comparisons were used to compare diagnostic categories. FINDINGS: The kappas for the three readings of the overall data set using eight-category World Health Organization (WHO) criteria were as follows: 0.66 for the generalized, 0.72 for weighted, and ranged from 0.59 to 0.94 unweighted binary categories; those read using four-category Bethesda criteria: 0.70 for generalized, 0.69 for weighted, and 0.56-0.94 for unweighted binary categories. For the pool versus the study pathologist readings, the eight-category kappa was 0.51 for generalized, 0.72 for weighted, and 0.56-0.82 for unweighted binary categories; for those read using Bethesda criteria: 0.70 for generalized, 0.70 for weighted, and 0.59-0.82 for the unweighted binary categories. The interpathologist and intrapathologist readings were fair by Landis standards at the low end of the diagnostic scale (atypia, human papillomavirus, and CIN1) and substantial to almost perfect at the high end (CIN2, CIN3, and CIS). The randomly selected and randomly ordered sample of 117 specimens read with the WHO system yielded a generalized kappa of 0.45; among the three institutions (M. D. Anderson Cancer Center vs. BCCA, M. D. Anderson vs. BWH, and BCCA vs. BWH), the unweighted kappas were 0.46, 0.41, and 0.49 and the weighted were 0.65, 0.66, and 0.68, respectively; for the Bethesda, a generalized kappa of 0.65, unweighted kappas of 0.66, 0.65, and 0.47, and weighted of 0.74, 0.72, and 0.74. The difficult specimens read with the WHO system yielded a generalized kappa of 0.23; among the three institutions the unweighted kappas were 0.20, 0.30, and 0.37, and the weighted were 0.17, 0.34, and 0.31; for the Bethesda, a generalized kappa of 0.25; among the three institutions, the unweighted kappas were 0.21, 0.32, and 0.37, and the weighted were: 0.07, 0.21, and 0.37, respectively. INTERPRETATION: Kappas in this expert group of pathologists were in the moderate, substantial, and almost perfect ranges for the overall and randomized samples. The randomized sample was representative of the larger sample. The kappa of the specimens for which disagreements arose was, predictably, in the slight range. Our findings will aid both the correlations with optical measurements using fluorescence and reflectance spectroscopy and the quantitative histopathologic analysis of these study specimens.

Confocal DNA cytometry: a contour-based segmentation algorithm for automated three-dimensional image segmentation.

BACKGROUND: Confocal laser scanning microscopy (CLSM) presents the opportunity to perform three-dimensional (3D) DNA content measurements on intact cells in thick histological sections. So far, these measurements have been performed manually, which is quite time-consuming. METHODS: In this study, an intuitive contour-based segmentation algorithm for automatic 3D CLSM image cytometry of nuclei in thick histological sections is presented. To evaluate the segmentation algorithm, we measured the DNA content and volume of human liver and breast cancer nuclei in 3D CLSM images. RESULTS: A high percentage of nuclei could be segmented fully automatically (e.g., human liver, 92%). Comparison with (time-consuming) interactive measurements on the same CLSM images showed that the results were well correlated (liver, r = 1.00; breast, r = 0.92). CONCLUSIONS: Automatic 3D CLSM image cytometry enables measurement of volume and DNA content of large numbers of nuclei in thick histological sections within an acceptable time. This makes large-scale studies feasible, whereby the advantages of CLSM can be exploited fully. The intuitive modular segmentation algorithm presented in this study detects and separates overlapping objects, also in two-dimensional (2D) space. Therefore, this algorithm may also be suitable for other applications.