RNA-sequencing predicts a role of androgen receptor and aldehyde dehydrogenase 1A1 in osteosarcoma lung metastases.

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM), which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65-70%, patients with LM experience poor survival rates of only 19-30%. Unacceptably, this situation that has remained unchanged for 30 years. Thus, there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors, including 4 matched sets, were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy.

Grade 2, 3 and Dedifferentiated Chondrosarcomas: A Comparative Study of Isocitrate Dehydrogenase-Mutant and Wild-Type Tumors with Implications for Prognosis and Therapy.

BACKGROUND: Grade 2 and 3 and dedifferentiated chondrosarcomas (CS) are frequently associated with isocitrate dehydrogenase (IDH) mutations and often exhibit a poor clinical outcome. Treatment is limited mainly to surgery. Defining IDH status (wild type (WT) and mutant) and the associated transcriptome may prove useful in determining other therapeutic options in these neoplasms. METHODS: Formalin-fixed paraffin-embedded material from 69 primary and recurrent grade 2, 3 and dedifferentiated CS was obtained. DNA sequencing for IDH1 and IDH2 mutations (n = 47) and RNA sequencing via Nextseq 2000 (n = 14) were performed. Differentially expressed genes (DEGs) were identified and used to predict aberrant biological pathways with Ingenuity Pathway Analysis (IPA) software (Qiagen). Gene Set Enrichment Analyses (GSEA) using subsets C3, C5 and C7 were performed. Differentially expressed genes were validated by immunohistochemistry. Outcome analysis was performed using the Wilcoxon test. RESULTS: A set of 69 CS (28 females, 41 males), average age 65, distributed among femur, pelvis, humerus, and chest wall were identified from available clinical material. After further selection based on available IDH status, we evaluated 15 IDH WT and 32 IDH mutant tumors as part of this dataset. Out of 15 IDH WT tumors, 7 involved the chest wall/scapula, while 1 of 32 mutants arose in the scapula. There were far more genes overexpressed in IDH WT tumors compared to IDH mutant tumors. Furthermore, IDH WT and IDH mutant tumors were transcriptomically distinct in the IPA and GSEA, with IDH mutant tumors showing increased activity in methylation pathways and endochondral ossification, while IDH WT tumors showed more activity in normal matrix development pathways. Validation immunohistochemistry demonstrated expression of WT1 and AR in IDH WT tumors, but not in IDH mutants. SATB2 was expressed in IDH mutant tumors and not in WT tumors. Outcome analysis revealed differences in overall survival between mutant and WT tumors (p = 0.04), dedifferentiated mutant and higher-grade (2, 3) mutant tumors (p = 0.03), and dedifferentiated mutant and higher-grade (2, 3) WT tumors (p = 0.03). The longest survival times were observed in patients with higher-grade WT tumors, while patients with dedifferentiated mutant tumors showed the lowest survival. Generally, patients with IDH WT tumors displayed longer survival in both the higher-grade and dedifferentiated groups. CONCLUSIONS: Grade 2, 3 and dedifferentiated chondrosarcomas are further characterized by IDH status, which in turn informs transcriptomic phenotype and overall survival. The transcriptome is distinct depending on IDH status, and implies different treatment targets.

WCRC-25: A novel luminal Invasive Lobular Carcinoma cell line model.

Breast cancer is categorized by the molecular and histologic presentation of the tumor, with the major histologic subtypes being No Special Type (NST) and Invasive Lobular Carcinoma (ILC). ILC are characterized by growth in a single file discohesive manner with stromal infiltration attributed to their hallmark pathognomonic loss of E-cadherin ( CDH1 ). Few ILC cell line models are available to researchers. Here we report the successful establishment and characterization of a novel ILC cell line, WCRC-25, from a metastatic pleural effusion from a postmenopausal Caucasian woman with metastatic ILC. WCRC-25 is an ER-negative luminal epithelial ILC cell line with both luminal and Her2-like features. It exhibits anchorage independent growth and haptotactic migration towards Collagen I. Sequencing revealed a CDH1 Q706* truncating mutation, together with mutations in FOXA1, CTCF, BRCA2 and TP53 , which were also seen in a series of metastatic lesions from the patient. Copy number analyses revealed amplification and deletion of genes frequently altered in ILC while optical genome mapping revealed novel structural rearrangements. RNA-seq analysis comparing the primary tumor, metastases and the cell line revealed signatures for cell cycle progression and receptor tyrosine kinase signaling. To assess targetability, we treated WCRC-25 with AZD5363 and Alpelisib confirming WCRC-25 as susceptible to PI3K/AKT signaling inhibition as predicted by our RNA sequencing analysis. In conclusion, we report WCRC-25 as a novel ILC cell line with promise as a valuable research tool to advance our understanding of ILC and its therapeutic vulnerabilities. Financial support: The work was in part supported by a Susan G Komen Leadership Grant to SO (SAC160073) and NCI R01 CA252378 (SO/AVL). AVL and SO are Komen Scholars, Hillman Foundation Fellows and supported by BCRF. This project used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource which is supported in part by award P30CA047904. This research was also supported in part by the University of Pittsburgh Center for Research Computing, RRID:SCR_022735, through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. Finally, partial support was provided by the Magee-Womens Research Institute and Foundation, The Shear Family Foundation, and The Metastatic Breast Cancer Network.

Intraoperative Evaluation of Soft Tissue Sarcoma Surgical Margins with Indocyanine Green Fluorescence Imaging.

Soft tissue sarcomas (STS) are rare malignant tumors often associated with poor outcomes and high local recurrence rates. Current tools for intraoperative and definitive margin assessment include intraoperative frozen section and permanent pathology, respectively. Indocyanine green dye (ICG) is a historically safe fluorophore dye that has demonstrated efficacy for intraoperative margin assessment in the surgical management of both breast and gastrointestinal cancers. The utility of ICG in the surgical management of sarcoma surgery has primarily been studied in pre-clinical mouse models and warrants further investigation as a potential adjunct to achieving negative margins. This study is a prospective, non-randomized clinical study conducted on patients with confirmed or suspected STS. Patients younger than 18 years, with a prior adverse reaction to iodine or fluorescein, or with renal disease were excluded from the study. Intravenous ICG was infused approximately three hours prior to surgery at a dosage of 2.0-2.5 mg/kg, and following tumor resection, the excised tumor and tumor bed were imaged for fluorescence intensity. When scanning the tumor bed, a threshold of 77% calibrated to the region of maximum intensity in the resected tumor was defined as a positive ICG margin, according to published protocols from the breast cancer literature. ICG results were then compared with the surgeon's clinical impression of margin status and permanent pathology results. Out of 26 subjects recruited for the original study, 18 soft tissue sarcomas (STS) were included for analysis. Three subjects were excluded for having bone sarcomas, and five subjects were excluded due to final pathology, which was ultimately inconsistent with sarcoma. The average age of patients was 64.1 years old (range: 28-83), with an average ICG dose of 201.8 mg. In 56% (10/18) of patients, ICG margins were consistent with the permanent pathology margins, with 89% specificity. The use of ICG as an intraoperative adjunct to obtaining negative margins in soft tissue sarcoma surgery is promising. However, studies with larger sample sizes are warranted to further delineate the accuracy, optimal dosage, timing, and types of sarcoma in which this diagnostic tool may be most useful.

Patient-Derived Spheroid Culture Models Are Better Than Monolayer Models in Chondrosarcoma Research.

Purpose: Chondrosarcoma (CSA) are mesenchymal tissue-derived bone tumors. CSA mainly occurs in older people. CSA has demonstrated resistance to chemotherapy and radiation; complete surgical removal with negative margins is the only treatment option. In the case of metastatic CSA, the chance of survival is meager. Since the conventional two-dimensional cell culture models failed to retain tumor characteristics, developing preclinical models mimicking the disease with the highest fidelity is paramount for personalized treatments. Methods: In this study, we established spherical cultured cells as new models for CSA. First, we demonstrated that CSA cells could form spheroids when cultured in ultra-low attachment plates. Next, tissue samples from CSA patients were collected and processed into primary cells, which were subsequently cultured as primary spheroids. The growth rate of primary spheroids was monitored and the histology of mature spheroids were characterized. These primary spheroids were used in drug susceptibility studies where traditional doxorubicin therapy and our novel disulfiram-copper therapy were tested. Results: Compared with conventional monolayer cultures, spheroids better recapitulated the features of the in vivo tumor in the aspect of the formation of extracellular matrix. In the drug susceptibility study, spheroids demonstrated high resistance to the classic therapies, suggesting that monolayer cultures may give false positive results. Therefore, using spheroids for drug research and development in the CSA field should provide more accurate results. Conclusion: In summary, our study of primary CSA spheroids brought new insight into their chemoresistance and demonstrated its potential for personalized treatment of CSA in clinical medicine.

Prognostic factors and survival of patients undergoing surgical intervention for breast cancer bone metastases.

INTRODUCTION: Bone is the most common distant site of breast cancer metastasis. Skeletal lesions can cause significant morbidity due to pain, pathologic fracture, and electrolyte abnormalities. Current treatment for patients with bone metastases (BoM) from breast cancer is highly personalized and often involves a multidisciplinary approach with chemotherapy, hormone therapy, bone-targeted antiresorptive agents, radiation therapy, and surgery. We have retrospectively collected clinical data from a series of patients with bone metastases to evaluate the clinical characteristics, prognostic factors, and survival patterns of patients with breast cancer BoM receiving standard multimodal therapy. METHODS: A consecutive series of 167 patients with breast cancer BoM treated at a single institution between August 2013 and March 2020 were identified. Clinical information was obtained from the medical record and survival analyses were performed to evaluate patient outcomes and identify prognostic factors. RESULTS: Thirty-seven patients (22%) presented with de novo BoM - bone metastases at the time of breast cancer diagnosis - and were 2.6 times more likely to die within the study period than those with asynchronous BoM (HR = 2.62, p = <0.0001). Patients who received bone-targeted medical therapy were 61% less likely to die after BoM diagnosis than those who did not (HR = 0.39, p = 0.001). Operative stabilization of BoM was more frequently employed in patients with lytic (p = 0.02) or mixed (p = 0.02) tumors than it was for those with blastic lesions. Patients treated with surgery had a lower overall bone metastasis survival than those treated without (p < 0.03). DISCUSSION: These findings reflect the current patterns in metastatic breast cancer treatment and associated outcomes. In a series of 167 consecutive patients, we demonstrate the natural history of breast cancer with BoM being treated with modern multimodal therapy. Understanding these treatment patterns and prognostic factors enhances the provider's ability to counsel patients and direct appropriate treatments.

Do Patient-derived Spheroid Culture Models Have Relevance in Chondrosarcoma Research?

BACKGROUND: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor's biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described. QUESTIONS/PURPOSES: (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments? METHODS: Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader. RESULTS: Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p < 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p < 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) < 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) < 0.0001) and tumors (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment. CONCLUSION: We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted. CLINICAL RELEVANCE: Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.