Immortalization and transformation of primary cells mediated by engineered ecDNAs.

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells. We demonstrate the feasibility of this approach by engineering MDM2-containing ecDNAs in near-diploid human cells, showing that GFP expression can be used to track ecDNA dynamics under physiological conditions or in the presence of specific selective pressures. We also apply this approach to generate mice harboring inducible Myc - and Mdm2 -containing ecDNAs analogous to those spontaneously occurring in human cancers. We show that the engineered ecDNAs rapidly accumulate in primary cells derived from these animals, promoting proliferation, immortalization, and transformation.

Feasibility of Preoperative Dose-Painting Intensity Modulated Radiation Therapy (IMRT) for Borderline-Resectable Primary Retroperitoneal Sarcoma.

PURPOSE: The role of radiation therapy (RT) in resectable retroperitoneal sarcoma (RPS) remains controversial; however, preoperative RT may play an important role in borderline-resectable (at risk of R2 resection) disease. We evaluated the outcome of such patients treated with preoperative dose-painting IMRT followed by planned resection. METHODS: Between January 2001 and December 2017, 30 patients with borderline-resectable primary nonmetastatic RPS (after multidisciplinary review) received preoperative dose-painting IMRT in this retrospective cohort study. RESULTS: Median follow-up for all patients was 32 months. Median dose to the whole tumor/high-risk margin was 50.4 Gy/60.2 Gy. Sixteen patients were female, 24 were >50 years. Median tumor size was 9.2 cm. After RT, 6 did not have surgery. Of the 24 who were explored, 20 underwent complete gross resection. During RT, 7 of 30 patients developed acute grade 2+ toxicities: 5 fatigue, 1 nausea and vomiting, and 1 cystitis. RT was completed in 29 of 30 patients. Postoperatively, 12 of 20 patients developed grade 2+ complications: 2 gastropathy, 5 intraabdominal collections requiring drainage, 1 retroperitoneal bleed, and 3 delayed wound healing. Late grade 2+ toxicity was observed in 3 of 20 patients: 1 lymphedema with recurrent cellulitis, 1 chronic diarrhea, 1 gastrointestinal bleeding from anastomosis requiring transfusions, and 2 renal insufficiency. In those who underwent complete gross resection (n = 20, median follow-up 47 months), the 5-year local control was 57%, and overall survival was 46%. DISCUSSION: Preoperative dose-painting IMRT given to borderline-resectable RPS rendered 67% of patients resectable, provided a 5-year local control rate of 57%, which is similar to those with resectable disease, and had an acceptable morbidity profile.

Simulations of goaf surface subsidence via filling control.

Xincheng Gold Mine is taken as an example to investigate the ground subsidence that results from filling. Both numerical simulations and simulation experiments are conducted to simulate the deformation process at the stope roof and bottom from excavation and filling. The assumption of macroscopic continuity from traditional continuum mechanics models is overcome. The simulation results demonstrate that the ground subsidence is slowed due to filling. The total trends of the top and bottom displacements are sinkable and upturned, respectively. Moreover, with an increased buried depth and lateral pressure coefficient, the displacements of the top and bottom of the stope increase as well. The characteristics and evolution of the displacement vector field of the rock mass are macroscopically and microscopically studied over the excavation progress. This provides technical support for stope safety production.

Radiation Therapy in Primary Soft Tissue Sarcoma of the Superficial Trunk.

PURPOSE: Despite anatomical differences, truncal soft tissue sarcomas (STS) often are grouped with extremity sarcomas. We evaluated the clinical outcome of patients with truncal STS who underwent gross total resection (GTR) and radiation therapy (RT), with special emphasis on those treated with intensity modulated radiation therapy (IMRT). METHODS: From January 1, 2001 to December 31, 2018, 64 patients received GTR and RT, where 48 patients were male, 35 patients were aged ≤ 60 years, and 48 patients had tumors ≤ 10 cm. Sixty-two tumors were high grade, 36 were in the chest wall, 7 in the abdominal wall, and 21 were paraspinal. During surgery, 7 received mesh reconstruction, and 6 received flap closure. R0 resection was achieved in 53 patients. Thirteen patients received chemotherapy. RESULTS: With a median follow-up of 57 months, the 5-year actuarial local control (LC) was 71%. In the IMRT subset (50/64, 78%), the 5-year LC for the chest/abdominal wall was 84%, and 69% for the paraspinal subsite. Grade 2+ radiation dermatitis was seen in 21 of 64 (33%) patients, 5 of 64 (8%) developed noninfectious wound complications, 5 of 64 (8%) developed infectious wound complications, and 1 of 64 (2%) developed grade 2 chest wall pain. No additional grade 2+ late toxicity was observed. CONCLUSIONS: Based on this study, achieving LC in truncal STS treated with GTR and RT remains challenging even with IMRT (5-year LC: 78%). While the use of IMRT was more promising for tumors of the chest/abdominal wall with 5-year LC of 84%, it was 69% for those located in the paraspinal subsite, indicating a need for further improvement.

Combined Compressed Sensing and SENSE to Enhance Radiation Therapy Magnetic Resonance Imaging Simulation.

PURPOSE: To assess the effect of a combination of compressed sensing and SENSitivity Encoding (SENSE) acceleration techniques on radiation therapy magnetic resonance imaging (MRI) simulation workflows. METHODS AND MATERIALS: Thirty-seven acquisitions were performed with both SENSE-only (SENSE) and combined compressed sensing and SENSE (CS) techniques in 24 patients receiving radiation therapy MRI simulation for a wide range of disease sites. The anatomic field of view prescription and image resolution were identical for both SENSE and CS acquisitions to ensure fair comparison. The acquisition time of all images was recorded to assess time savings. For each image pair, image quality, and ability to contour were assessed by 2 radiation oncologists. Aside from direct image pair comparisons, the feasibility of using CS to improve MRI simulation protocols by increasing image resolution, field of view, and reducing motion artifacts was also evaluated. RESULTS: CS resulted in an average reduction of 27% in scan time with negligible changes in image quality and the ability to contour structures for RT treatment planning compared with SENSE. Physician scoring of image quality and ability to contour shows that while SENSE still has slightly better image quality compared with CS, this observed difference in image quality did not affect the ability to contour. In addition, the higher acceleration capability of CS enabled use of superior-inferior direction phase encoding in a sagittal 3-dimensional T2-weighted scan for substantially improved visibility of the prostatic urethra, which eliminated the need for a Foley catheter in most patients. CONCLUSIONS: The combination of compressed sensing and parallel imaging resulted in marked improvements in the MRI Simulation workflow. The scan time was reduced without significantly affecting image quality in the context of ability to contour. The acceleration capabilities allowed for increased image resolution under similar scanning times as well as significantly improved urethra visualization in prostate simulations.

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo.

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

Incidence and Dosimetric Predictors of Radiation-Induced Gastric Bleeding After Chemoradiation for Esophageal and Gastroesophageal Junction Cancer.

PURPOSE: To determine the incidence and predictors of gastric bleeding after chemoradiation for esophageal or gastroesophageal junction cancer. METHODS AND MATERIALS: We reviewed patients receiving chemoradiation to at least 41.4 Gy for localized esophageal cancer whose fields included the stomach and who did not undergo surgical resection. The primary endpoint was grade ≥3 gastric hemorrhage (GB3+). Comprehensive stomach dose-volume parameters were collected, and stomach dose-volume histograms were generated for analysis. RESULTS: A total of 145 patients met our inclusion criteria. Median prescribed dose was 50.4 Gy (range, 41.4-56 Gy). Median stomach Dmax was 53.0 Gy (1.0-62.7 Gy), and median stomach V40, V45, and V50 Gy were 112 cm3 (0-667 cm3), 84 cm3 (0-632 cm3), and 50 cm3 (0-565 cm3), respectively. Two patients (1.4%) developed radiation-induced GB3+. The only dosimetric factor that was significantly different for these patients was a higher stomach Dmax (58.1 and 58.3 Gy) than the cohort median (53 Gy). One of these patients also had cirrhosis, and the other had a history of nonsteroidal anti-inflammatory drug use. Five other patients had GB3+ events associated with documented tumor progression. A Cox proportional hazards model based on stomach Dmax with respect to the development of GB3+ was found to be statistically significant. Time-to-event curves and dose-volume atlases were generated, demonstrating an increased risk of GB3+ only when stomach Dmax was >58 Gy (P < .05). CONCLUSIONS: We observed a low rate of GB3+ events in patients who received chemoradiation to a median dose of 50.4 Gy to volumes that included a significant portion of the stomach. These results suggest that when prescribing 50.4 Gy for esophageal cancer, there is no need to minimize the irradiated gastric volume or dose for the sake of preventing bleeding complications. Limiting stomach maximum doses to <58 Gy may also avoid bleeding, and particular caution should be taken in patients with other risk factors for bleeding, such as cirrhosis.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Comparison of outcomes in early-stage uterine clear cell carcinoma and serous carcinoma.

PURPOSE: The treatment paradigm for uterine clear cell carcinoma is often linked to serous carcinoma. This study compares oncologic outcomes between women with uterine clear cell and serous carcinoma. METHODS AND MATERIALS: We reviewed 114 women with stage I-II uterine clear cell carcinoma (n = 17, 15%) or serous carcinoma (n = 97, 85%) who underwent hysterectomy and salpingo-oophorectomy at our institution from April 1992 to December 2011; 86 (76%) had stage IA, 14 (12%) had stage IB, and 14 (12%) had stage II disease. Median followup was 57 months. RESULTS: Patients with uterine clear cell and serous carcinoma did not differ significantly by age ≥60 years, stage, or rate of lymphovascular invasion. There was no difference in the number of patients with clear cell or serous histology who received adjuvant radiotherapy (71% vs. 84%, respectively; p = 0.31); however, significantly fewer patients with clear cell histology received adjuvant chemotherapy (35% vs. 67%, respectively; p = 0.02). At 5 years, there were no significant differences in disease-free survival (94% vs. 84%, respectively; p = 0.27), disease-specific survival (100% vs. 92%, respectively; p = 0.20), or overall survival (100% vs. 89%, respectively; p = 0.34). The differences in chemotherapy utilization did not impact pattern of relapse, specifically peritoneal spread (7% vs. 6%, respectively; p = 0.92) or other distant sites (0% vs. 9%, respectively; p = 0.17). CONCLUSIONS: Oncologic outcomes and recurrence patterns of women with stage I-II uterine clear cell carcinoma compared favorably with those of women with serous carcinoma, despite significantly less adjuvant chemotherapy use. Potential reduction in adjuvant therapy in women with clear cell carcinoma should be studied prospectively.

Radiation techniques for esophageal cancer.

Radiotherapy plays a crucial role in the curative management of localized esophageal cancer, both as definitive and preoperative therapy. For definitive therapy, the standard radiation dose is 50.4 Gy in 28 fractions and should be delivered with concurrent chemotherapy. Chemoradiotherapy also has a wellestablished benefit in the preoperative setting, as established in the CROSS randomized trial. Radiation fields are typically generous, to account for subclinical extension of disease along the esophagus and to regional nodes. Three-dimensional conformal radiation is the current standard technique for esophageal cancer, though intensity-modulated radiation therapy is increasingly utilized and may improve the outcomes of esophageal radiotherapy by reducing radiation dose to critical normal tissues.

The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis.

Novel genetically engineered mouse models using the Cre-loxP or the Flp-FRT systems have generated useful reagents to manipulate the mouse genome in a temporally-regulated and tissue-specific manner. By incorporating a constitutive Cre driver line into a mouse model in which FRT-regulated genes in other cell types are regulated by Flp-FRT recombinase, gene expression can be manipulated simultaneously in separate tissue compartments. This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy. Generating mice in which Cre-ER(T2) is expressed under Flp-FRT-mediated regulation would enable step-wise manipulation of the mouse genome using dual recombinase technology. Such next-generation mouse models would enable sequential mutagenesis to better model cancer and define genes required for tumor maintenance. Here, we generated novel genetically engineered mice that activate or delete Cre-ER(T2) in response to Flp recombinase. To potentially utilize the large number of Cre-loxP-regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ER(T2) alleles into the endogenous Col1a1 locus for ubiquitous expression. In the Col1a1(FRT-Cre-ER-T2-FRT) mice, Flp deletes Cre-ER(T2), so that Cre-ER(T2) is only expressed in cells that have never expressed Flp. In contrast, in the Col1a1(FRT-STOP-FRT-Cre-ER-T2) mice, Flp removes the STOP cassette to allow Cre-ER(T2) expression so that Cre-ER(T2) is only expressed in cells that previously expressed Flp. These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis and cancer.

HIF-1 Alpha Regulates the Response of Primary Sarcomas to Radiation Therapy through a Cell Autonomous Mechanism.

Hypoxia is a major cause of radiation resistance, which may predispose to local recurrence after radiation therapy. While hypoxia increases tumor cell survival after radiation exposure because there is less oxygen to oxidize damaged DNA, it remains unclear whether signaling pathways triggered by hypoxia contribute to radiation resistance. For example, intratumoral hypoxia can increase hypoxia inducible factor 1 alpha (HIF-1α), which may regulate pathways that contribute to radiation sensitization or radiation resistance. To clarify the role of HIF-1α in regulating tumor response to radiation, we generated a novel genetically engineered mouse model of soft tissue sarcoma with an intact or deleted HIF-1α. Deletion of HIF-1α sensitized primary sarcomas to radiation exposure in vivo. Moreover, cell lines derived from primary sarcomas lacking HIF-1α, or in which HIF-1α was knocked down, had decreased clonogenic survival in vitro, demonstrating that HIF-1α can promote radiation resistance in a cell autonomous manner. In HIF-1α-intact and -deleted sarcoma cells, radiation-induced reactive oxygen species, DNA damage repair and activation of autophagy were similar. However, sarcoma cells lacking HIF-1α had impaired mitochondrial biogenesis and metabolic response after irradiation, which might contribute to radiation resistance. These results show that HIF-1α promotes radiation resistance in a cell autonomous manner.

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes.

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.

RAS and ROS in rhabdomyosarcoma.

The 5-year survival for localized rhabdomyosarcoma is over 70%, but only 30% for patients presenting with metastatic disease. In this issue of Cancer Cell, Chen and colleagues performed whole-genome and RNA sequencing on human rhabdomyosarcoma and identified RAS mutations and oxidative stress as potential therapeutic targets for high-risk embryonal rhabdomyosarcoma.

Distinct and overlapping sarcoma subtypes initiated from muscle stem and progenitor cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis.

UNLABELLED: Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE: Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.