Developed Lung Metastases Research Articles

β-glucan from Lentinus edodes inhibits breast cancer progression via the Nur77/HIF-1α axis.

Background: β-glucan from Lentinus edodes (LNT) is a plant-derived medicinal fungus possessing significant bioactivities on anti-tumor. Both hypoxia-induced factor-1α (HIF)-1α and Nur77 have been shown to be involved in the development of breast cancer. However, there is yet no proof of Nur77/HIF-1α involvement in the process of LNT-mediated tumor-inhibition effect.Methods: Immunohistochemistry, immunofluorescence and Hematoxylin–Eosin staining were used to investigate tumor growth and metastasis in MMTV-PyMT transgenic mice. Proliferation and metastasis-associated molecules were determined by Western blotting and reverse transcription-quantitative PCR. Hypoxic cellular model was established under the exposure of CoCl2. Small interference RNA was transfected using Lipofectamine reagent. The ubiquitin proteasome pathway was blunted by adding the proteasome inhibitor MG132.Results: LNT inhibited the growth of breast tumors and the development of lung metastases from breast cancer, accompanied by a decreased expression of HIF-1α in the tumor tissues. In in vitro experiments, hypoxia induced the expression of HIF-1α and Nur77 in breast cancer cells, while LNT addition down-regulated HIF-1α expression in an oxygen-free environment, and this process was in a manner of Nur77 dependent. Mechanistically, LNT evoked the down-regulation of HIF-1α involved the Nur77-mediated ubiquitin proteasome pathway. A strong positive correlation between Nur77 and HIF-1α expression in human breast cancer specimens was also confirmed.Conclusion: Therefore, LNT appears to inhibit the progression of breast cancer partly through the Nur77/HIF-1α signaling axis. The findings of the present study may provide a theoretical basis for targeting HIFs in the treatment of breast cancer.

Biomarker alterations associated with distinct patterns of metastatic spread in colorectal cancer

Metastatic spread is the most important life-threatening feature of colorectal cancer and is supposed to be mainly driven by alterations in different carcinogenic pathways. The present study compared mutation and expression profiles of distinctive biomarkers in colorectal cancer patients with different clinical metastatic patterns. As for a case-control study, patients were matched according to T category, grading and primary tumour site. Overall, 246 patients with either exclusive lung metastasis (N = 82), exclusive liver metastasis (N = 82) or non-metastatic colorectal cancer (N = 82) were identified. Paraffin-embedded specimens were examined for mutations in the RAS and RAF genes and for the expression of β-catenin and CD133. Clinical endpoints were presence or absence of distant metastasis, formation of metastasis in lungs versus the liver and survival. MAPK pathway mutations in either the KRAS, NRAS or BRAF gene were associated with the development of lung metastasis (63.4%) compared to the control group (47.6%; p = 0.04). MAPK pathway alterations plus high β-catenin expression were associated with metastasis to the lungs but not to the liver (28.0% vs. 13.4%; p = 0.02). High CD133 expression correlated with the development of liver metastasis compared to the control group (30.5% vs. 14.6%; p = 0.02). This data indicates that different patterns of distant spread are associated with specific biomarker alterations and may represent different molecular subtypes of colorectal cancer. However, underlying mechanisms of metastasis formation in different anatomic sites remains unclear. Since knowledge of the anticipated site of distant spread would substantially impact clinical management, further research is needed to identify solid biomarkers for different metastatic patterns.

Survival and treatment outcome of head and neck cancer patients with pulmonary oligometastases.

The purpose of this study was to determine the outcome of head and neck squamous cell carcinoma (HNSCC) patients developing lung metastasis. Retrospective study. HNSCC patients with lung metastasis treated between 2001 and 2018 were included. Statistical analyses described the relationship between patient survival, treatment efficacy and pulmonary metastasis occurrence. One hundred HNSCC patients were included in the study. The median overall survival (OS) was 21months. The median recurrence-free survival (RFS) was seven months. Patient survival with only lung metastases was significantly longer compared to patients with lung metastases and lymph nodes involvement or other metastases. Moreover, patients with a single metastasis had longer post-RFS and OS than patients with multiple metastases. The local control of metastasis was better when patients presented only lung metastases, and it was more effective in single metastasis. The surgery allowed better metastases local control than supportive care or radio and/or chemotherapy. In case of specific therapy, pulmonary resection was associated with a longer post-RFS and a longer OS compared to supportive care or radio and/or chemotherapy. We confirmed, in the current study, the significant survival benefit for HNSCC patients treated by surgery for their pulmonary metastasis. While treatment of multiple metastases required palliative chemotherapy or best supportive care in most of the cases, specific surgical treatment in selected HNSCC patients should be considered.

A Potential Role of Adhesion Molecules on Lung Metastasis Enhanced by Local Inflammation.

Local and systemic inflammations are associated with negative long-term outcomes; however, their precise mechanism of action remains unclear. We previously demonstrated that hepatocyte growth factor (HGF)/c-Met signaling contributed to the enhancement of liver metastasis associated with peritonitis model. The aim of this study is to investigate the effect of local inflammation on the development of lung metastasis. NL-17 cells were injected into BALB/c mice via the tail vein to produce a high potential model for lung metastasis. After injection of NL-17 cells, lipopolysaccharide (LPS) and live Pseudomonas aeruginosa, and phosphate-buffered saline were administered intratracheally to induce acute lung injury (ALI) and pneumonia, respectively. In both ALI and pneumonia mice, lung metastasis was significantly promoted compared to control mice. Concentrations of Interleukin-6, tumor necrosis factor-α, and HGF in the bronchoalveolar lavage fluid were significantly higher in ALI and pneumonia mice than in control mice. Neither administration of recombinant mouse HGF nor c-Met knockdown in NL-17 cells influenced the magnitude of lung metastasis. Yet stimulation with LPS increased the expression of α2 integrin, vascular cell-adhesion protein-1, and intercellular adhesion molecule-1 (ICAM-1) in the lung. Invasive activity of NL-17 cells was significantly up-regulated by LPS, but was suppressed by anti-ICAM-1 antibody. While LPS-stimulated NL-17 cells showed significantly promoted lung metastasis, E-selectin expression in the lungs of mice with ALI or pneumonia was significantly enhanced compared with control mice. Up-regulation of adhesion molecules, but not HGF/c-Met signaling, may contribute to the lung metastasis enhanced by local infection/inflammation.

The Long Noncoding RNA NEAT1 Promotes Sarcoma Metastasis by Regulating RNA Splicing Pathways.

Soft-tissue sarcomas (STS) are rare malignancies showing lineage differentiation toward diverse mesenchymal tissues. Half of all high-grade STSs develop lung metastasis with a median survival of 15 months. Here, we used a genetically engineered mouse model that mimics undifferentiated pleomorphic sarcoma (UPS) to study the molecular mechanisms driving metastasis. High-grade sarcomas were generated with Cre recombinase technology using mice with conditional mutations in Kras and Trp53 (KP) genes. After amputation of the limb bearing the primary tumor, mice were followed for the development of lung metastasis. Using RNA-sequencing of matched primary KP tumors and lung metastases, we found that the long noncoding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 (Neat1) is significantly upregulated in lung metastases. Furthermore, NEAT1 RNA ISH of human UPS showed that NEAT1 is upregulated within a subset of lung metastases compared with paired primary UPS. Remarkably, CRISPR/Cas9-mediated knockout of Neat1 suppressed the ability of KP tumor cells to colonize the lungs. To gain insight into the underlying mechanisms by which the lncRNA Neat1 promotes sarcoma metastasis, we pulled down Neat1 RNA and used mass spectrometry to identify interacting proteins. Interestingly, most Neat1 interacting proteins are involved in RNA splicing regulation. In particular, KH-Type Splicing Regulatory Protein (KHSRP) interacts with Neat1 and is associated with poor prognosis of human STS. Moreover, depletion of KHSRP suppressed the ability of KP tumor cells to colonize the lungs. Collectively, these results suggest that Neat1 and its interacting proteins, which regulate RNA splicing, are involved in mediating sarcoma metastasis. IMPLICATIONS: Understanding that lncRNA NEAT1 promotes sarcoma metastasis, at least in part, through interacting with the RNA splicing regulator KHSRP may translate into new therapeutic approaches for sarcoma.

The aryl hydrocarbon receptor promotes inflammation-induced dedifferentiation and systemic metastatic spread of melanoma cells.

The aryl hydrocarbon receptor (AHR) is a ligand binding-transcription factor of the basic helix-loop-helix family regulating multiple cellular functions such as differentiation, cell cycle, apoptosis, and inflammatory reactions. In neoplastic diseases, the AHR has been described to modulate proliferation and differentiation in dichotomous ways, either inhibiting or augmenting the growth of tumors. The precise role of AHR in melanoma is mostly unknown. Here, we report a functional effect of AHR activation on inflammation-induced melanoma cell dedifferentiation and the development of lung metastases in a mouse model. Via in silico analyses of "The Cancer Genome Atlas" human melanoma cohort, we detected a correlation between AHR expression levels and a dedifferentiated melanoma cell phenotype with an invasive gene signature, which we were able to functionally recapitulate in a panel of human melanoma cell lines. Both human and mouse melanoma cell lines upregulated AHR expression after inflammatory stimulation with tumor necrosis factor-α (TNF-α). Activation of AHR in human and mouse melanoma cell lines with the endogenous ligand formylindolo(3,2-b)carbazole (FICZ) promoted inflammation-induced dedifferentiation in vitro. Importantly, mouse melanoma cells with CRISPR/Cas9-mediated disruption of the AHR gene showed impaired in vivo tumor growth after transplantation in the skin as well as decreased numbers of spontaneous lung metastases. Taken together, our results demonstrate a functional role for AHR expression in melanoma development and metastatic progression. This provides a scientific basis for future experiments that further dissect the underlying molecular mechanisms and assess the potential for AHR inhibition as part of multimodal melanoma treatment strategies.

Development of lung metastases in mouse models of tongue squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) represents 3%-4% of all cancers. Despite the increasing incidence of OSCC distant metastasis and poor prognosis, few animal models of OSCC distant metastasis have been reported. In this study, we established mouse models of OSCC lung metastasis by orthotopic and tail vein injection of new OSCC cell lines. For the tail vein model, we used a novel cell line isolated from lung metastases reproduced in vivo after intravenous injection of HSC-3 GFP/luciferase cells and sorted for GFP expression (HSC-3 M1 GFP/luciferase). Lung metastases were assessed by imaging techniques and further confirmed by histology. For the orthotopic model, HSC-3 GFP/luciferase cells were injected into the tongue of athymic nude mice. The primary tumor and metastases were assessed by in vivo imaging, histology, and immunohistochemistry. The orthotopic model presented spontaneous lung metastases in 50% of the animals and lymph node metastases were present in 83% of cases. In the tail vein model, a lung metastasis rate of 60% was observed. Lung metastases were successfully reproduced by orthotopic and tail vein injection. Since lymph node metastases were present, the orthotopic model with HSC-3 GFP/luciferase cells may be suitable to investigate metastatic dissemination in OSCC.

Abstract 1474: IDO1 signaling supports inflammatory neovascularization

Abstract We have identified a distinct role for the immunoregulatory, tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase) in supporting neovascularization through its positioning as a key regulatory node between the inflammatory cytokines IFNγ (interferon γ) and IL6 (interleukin 6). IFNγ is a primary inducer of IDO1, but is also a key mediator of immune-based tumor suppression, which some studies have associated with its anti-angiogenic activity. Conversely, genetic studies in mice have clearly established a tumor-promoting role for IDO1, suggesting that it may interact with IFNγ in a negative feedback capacity. Targeted disruption of the Ido1 gene in mice resulted in enhanced resistance to lung tumor and metastasis development. This corresponded with attenuated induction of the pro-angiogenic cytokine IL6, which, when provided through ectopic expression, was able to restore pulmonary metastasis susceptibility to Ido1-/- mice. These initial findings suggested that IDO1 might contribute to cancer promotion by countering the anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of IL6. Consistent with this hypothesis, mice lacking either IDO1 or IL6 exhibited pulmonary metastasis resistance that was dependent on IFNγ and coupled with reduced tumor neovascularization. Neovascularization in a mouse oxygen-induced retinopathy model was likewise affected by IDO1 or IL6 loss, separating this process from the contextual complexity of the tumor microenvironment. Investigation into the cellular and molecular basis for the observed biology revealed that, within the heterogeneous expanse of Gr1+ MDSCs (myeloid-derived suppressor cells), the ability to independently elicit neovascularization in vivo was restricted to a highly autofluorescent population of CD11blo cells. This included a discrete, IDO1-expressing subpopulation, designated IDVCs (IDO1-dependent vascularizing cells), that, in an IDO1-dependent fashion, dominantly determined whether neovascularization was sustained. Mechanistically, the induction of IDO1 in IDVCs was found to provide a negative feedback constraint on the anti-angiogenic effect of host IFNγ through GCN2 (general control nonderepressible 2)-mediated activation of the integrated stress response and potentiation of IL6 production within these cells. These findings reveal fundamental molecular and cellular insights into how IDO1 interfaces with the inflammatory milieu to support neovascularization. Citation Format: Souvik Dey, Arpita Mondal, James B. DuHadaway, Erika Sutanto-Ward, Lisa Laury-Kleintop, Sunil Thomas, George C. Prendergast, Laura Mandik-Nayak, Alexander J. Muller. IDO1 signaling supports inflammatory neovascularization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1474.

Abstract 1823: Translating Myc inhibition to the clinic in metastatic breast cancer

Abstract Breast cancer is a leading cause of cancer mortality in women due to the high frequency of metastatic disease, which, despite advances in therapeutic options, is still essentially incurable. The role of Myc in promoting tumorigenesis is beyond doubt, but there are contradictory reports in the literature on its role in the metastatic process. Using a Myc dominant negative termed Omomyc, we have demonstrated in various mouse models that Myc inhibition is a safe and effective therapeutic approach against several types of cancer, regardless of the tissue of origin or the driver oncogenic lesion. So far, Omomyc has only been tested in primary tumors. However, since many steps of the metastatic cascade have been reported to depend on Myc, we hypothesized that Omomyc could be extremely effective in both the prevention and treatment of metastasis too. Here we show that Omomyc expression has a dramatic effect on colony formation capacity in human breast cancer cell lines representative of all the molecular subtypes of the disease. In MDA-MB-231 cells, not only did it impair their proliferation but also migration, invasion and their capacity to induce angiogenesis, key aspects of the metastatic process. We demonstrate that, in vivo, Omomyc reduces the growth of orthotopically-implanted human breast cancer cells in immunocompromised mice, induces regression of established metastases after primary tumor resection and impairs the development of lung metastases after tail vein injection. In the immunocompetent MMTV-PyMT transgenic model, Omomyc expression dramatically delays the formation and growth of mammary fat pad tumors, thereby preventing the appearance of lung metastases. When the purified Omomyc mini-protein is administered exogenously, we observe remarkable growth inhibition in vitro that recapitulates transgenic expression of Omomyc. Intravenous administration of the mini-protein reduces lung colonization and tumor growth in vivo. We have demonstrated for the first time the applicability of Omomyc against metastasis, challenging the pre-established notion that Myc inhibition could potentiate, rather than inhibit, invasion. Finally, we have validated the use of the purified Omomyc mini-protein as the first directly-deliverable Omomyc-based drug for the treatment of metastatic breast cancer, providing a new therapeutic opportunity for patients suffering from this dreadful and incurable disease. Citation Format: Daniel Massó-Vallés, Marie-Eve Beaulieu, Toni Jauset, Erika Serrano, Sandra Martínez-Martín, Laia Foradada, Virginia Castillo, Francisco Castillo, Génesis Martín, Sílvia Casacuberta-Serra, Mariano F. Zacarias-Fluck, Antonio Luque-García, Marta Escorihuela, Jonathan R. Whitfield, Joaquín Arribas, Laura Soucek. Translating Myc inhibition to the clinic in metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1823.

Comparison of the clinicopathological features between adenoid basal cell carcinoma and adenoid cystic carcinoma of the cervix

Objective: To compare the clinical and histopathological characteristics of cervical adenoid basal cell carcinoma and adenoid cystic carcinoma for improving the diagnosis accuracy and differential diagnosis of these tumors. Methods: A retrospective study was conducted on 9 cases of cervical adenoid basal cell carcinoma and 3 cases of adenoid cystic carcinoma which were diagnosed and consulted at the First Medical Center of PLA General Hospital from March 2009 to April 2019. Detailed clinical data were reviewed. All pathological sections and immunohistochemical results were reviewed and the clinicopathological characteristics were analyzed. Follow-up information by telephone was collected and relevant literature was consulted. Results: Both tumors were more commonly found in postmenopausal women (the age of onset ranged 43-74 years). Adenoid basal cell carcinoma was often clinical asymptomatic. Most of them presented as abnormal smears of the cervix during physical examination, and there was no definite mass in colposcopy.Adenoid cystic carcinoma was mostly presented with abnormal vaginal bleeding. A mass was seen in colposcopy.Histologically, the two tumors were characterized by nest-like growth of the tumors, consisting of basal-like tumor cells, and often surrounded by palisade structures. The two lesions might coexist, or be mixed with squamous cell carcinoma or high-grade squamous intraepithelial lesions. The difference was that adenoid basal cell carcinoma was mostly located at the junction of cervical squamous epithelium and columnar epithelium and beneath the overlying epithelium, the tumor cells were arranged in nests, with squamous differentiation in the center of the nests, or in double-layer adenoid arrangement. The cell morphology was bland with occasional mitoses, and the stromal reaction was not obvious. And adenoid cystic carcinoma cells in the nest arranged like a sieve, the homogenous red-stained and blue-stained secretions were observed in the sieve holes, with obvious cell atypia, frequent mitoses, and obvious stromal reaction.In one case of adenoid cystic carcinoma, sarcomatoid area presented around the nests.Both of them were positive in clinical HPV test. Among the 9 cases of adenoid basal cell carcinoma, 3 were tested for HPV and 5 were tested for p16, and all showed positive expression. Among the 3 cases of adenoid cystic carcinoma, 2 were tested for HPV and 3 were tested for p16, both of which showed positive expression. Telephone follow-up was conducted by June 2019(follow-up time ranged 2-37 months). No recurrence or metastasis occurred in 7 of the 9 cases of adenoid basal cell carcinoma, while 1 case had a ground-glass nodule in lung and another had recurrence of vaginal stump 32 months after the surgery.One case of adenoid cystic carcinoma developed lung metastasis 8 months after surgery and died 2 years after surgery; another case was followed up for 6 months, which showed no recurrence or metastasis; the third case was lost to follow-up. Conclusions: Both adenoid cystic carcinoma and adenoid basal cell carcinoma of the cervix are the tumors originating from cervical reserve cells and are associated with high-risk HPV infection. Due to the differences in clinical treatment and prognosis, careful histological evaluation and immunohistochemical analysis should be carried out to make accurate pathological diagnosis.

Abstract A07: Role of YAP/TEAD and YAP/Smad signaling pathways in osteosarcoma tumor growth and lung metastasis dissemination

Abstract Osteosarcoma (OS) is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because Hippo/YAP cascade plays a crucial role in cancer progression, we investigated its implication in OS development. Using proximity ligation assay and immunoprecipitation approaches, and promoter/reporter assays, we demonstrated a functional interaction between YAP and the transcriptional factor TEAD-1 in OS cell lines. To understand the specific role of this YAP/TEAD-1 interaction to regulate OS tumor growth, we generated OS cells that overexpressed a YAP protein able (YAP127A) or not able (YAP94A) to interact with TEAD-1. Using in vitro experiments and a preclinical model of OS, we demonstrated that YAP overexpression stimulates respectively OS cell proliferation and tumor growth only when YAP is able to interact with TEAD-1. Furthermore, the overexpression of YAPS94A reduces specific TEAD-1 transcriptional response and in vivo tumor growth. In contrast, the ability of YAP to stimulate OS cell migration does not depend on its ability to interact with TEAD-1. In addition, the effect of YAP overexpression (YAPS94A and YAPS127A) on OS cell migration is associated with cytoskeleton reorganization. Since we previously demonstrated the crucial role of the TGF-β/Smad3 signaling pathway in OS migration and metastatic development, we study the interaction between YAP and Smad3 in OS cells. Using proximity ligation assay and immunoprecipitation approaches, and promoter/reporter assays, we demonstrated a functional interaction between YAP and the transcriptional factor Smad3 in OS cell lines regardless of YAP’s ability to interact with TEAD-1. Finally, we investigated the effect of verteporfin, identified as an inhibitor of the Hippo signaling pathway, on OS progression. In vitro and in vivo experiments showed that verteporfin inhibits YAP/TEAD-1 cascade, OS cell proliferation, and reduces primary tumor growth. In addition, verteporfin reduces in vivo lung metastases development. In this context, in vitro experiments demonstrated that verteprofin inhibits the TGF-β/Smad3 cascade and the response of TGF-β key targets involved in the metastasis dissemination process. Together these results demonstrate that YAP/TEAD-1 interactions are crucial in OS tumor growth and suggest that YAP/SMAD3 interactions are involved in OS lung metastasis dissemination. Furthermore, we show that verteporfin may be a promising therapeutic strategy against tumor progression of OS, specifically against lung metastasis dissemination. Citation Format: Sarah Morice, Mathilde Mullard, Sarah Renault, Jérôme Amiaud, Régis Brion, Isabelle Corre, Anne Gomez-Brouchet, Françoise Redini, Franck Verrecchia. Role of YAP/TEAD and YAP/Smad signaling pathways in osteosarcoma tumor growth and lung metastasis dissemination [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A07.

The value of chest and skeletal staging in parosteal osteosarcoma: two-centre experience and literature review.

To determine the value of chest and skeletal staging in patients with parosteal osteosarcoma (POS), and the incidence of late local and chest recurrence. Retrospective review of patients across two institutions with histological confirmation of POS diagnosed between January 2007 and March 2020. Data collected included age, sex, skeletal location, results of chest CT and whole-body bone scintigraphy (WB-BSc) or whole-body MRI (WB-MRI) obtained at initial diagnosis. The histological tumour grade based on surgical resection specimens was classified as low-grade POS (LG-POS) and dedifferentiated POS (DD-POS). Findings of chest CT and skeletal staging were correlated with tumour grade. Follow-up chest CT and MRI studies were reviewed to determine the rate of late lung metastases and local recurrence. There were 27 males and 44 females, mean age 33years (range 12-79years). The femur (n = 43) and tibia (n = 14) were the most commonly involved bones. From surgical resection histology, 42 (59.2%) were LG-POS and 29 (40.8%) were DD-POS. WB-BSc/WB-MRI showed no skeletal metastases, while 1 case of DD-POS presented with bilateral calcified lung metastases. At follow-up, 7 patients (9.9%) developed lung metastases (mean of 18.9months, range 10-48months) from initial presentation, of which all were DD-POS. All but 1 patient who developed subsequent lung metastases had a local recurrence. Skeletal staging is unlikely to be of value in POS. Staging chest CT is very unlikely to demonstrate lung metastases in LG-POS and could be limited to DD-POS, particularly at the time of local recurrence.

Outcome of osteofibrous dysplasia-like versus classic adamantinoma of long bones: a single-institution experience

BackgroundThe clinical and molecular characteristics of osteofibrous dysplasia (OFD)-like adamantinoma (AD) differ from those of classic AD. Most reports about OFD-like AD are case reports or small case series. More cases from different centers are still warranted.MethodsThe orthopedic oncology database of our institution was searched to identify patients with AD. The cases of OFD-like and classic AD of the long bones were retrospectively analyzed. Between December 1999 and August 2016, 23 patients were treated for AD, comprising seven with OFD-like AD and 16 with classic AD. The outcomes were compared between AD subtypes.ResultsIn the OFD-like AD group, four lesions were treated with extensive curettage, while three were treated with wide resection. The median follow-up duration in the OFD-like AD group was 66 months (range 43–131 months). At the end of follow-up, there was only one case of local recurrence (LR) in the OFD-like AD group, giving a LR rate of 14.3% (1/7). No distant metastasis or progression to classic AD was detected in the OFD-like AD group. In the classic AD group, the treatments were below-the-knee amputation in one patient with extensive tibial and fibular lesions, curettage with a bone graft in one patient who was diagnosed with OFD based on a core needle biopsy, hemi-cortical excision and reconstruction in two patients, and segmental resection and reconstruction in 12 patients. At the end of follow-up, there were three cases of LR in the classic AD group, giving a LR rate of 18.8% (3/16); two patients developed lung metastasis after LR and died of the disease at 88 and 126 months after the first surgery in our hospital, respectively. The classic AD group had a metastatic rate of 12.5% (2/16), a final limb salvage rate of 75%, and estimated 5- and 10-year survival rates of 88.9% and 77.1%, respectively.ConclusionsOFD-like AD has a better outcome than classic AD. For OFD-like AD, extensive curettage is suggested if the tumor extent allows. For classic AD, aggressive resection with wide margins is essential to achieve local control. A long-term follow-up is necessary due to the possibility of late complications.

Abstract A29: Respective role of TEAD and Smad signaling in YAP-mediated osteosarcoma tumor growth and lung metastatic development

Abstract Osteosarcoma (OS) is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because a) gene set enrichment analysis (GSEA) of expression data from a cohort of OS patients reveals a Hippo conserved signature in OS and b) proximity ligation (PLA) and immunoprecipitation (IP) assays have shown an interaction between YAP and TEAD, the main effector of Hippo signaling pathway, in OS cell lines, we investigated its implication in OS development. To elucidate the role of TEAD in YAP-driven OS development, we probed the consequences of YAP activation using overexpression of YAPS127A (constitutively active YAP) and YAPS94A (TEAD-binding deficient YAP protein). Firstly, in vivo and in vitro experiments demonstrate a) the crucial role of TEAD in YAP-driven cell proliferation and in vivo tumor growth and b) a correlation between the expression of TEAD1 and the survival outcomes of OS patients. In this context RNAseq analysis and GSEA identified more than 300 genes related to cell proliferation that are significantly overexpressed in YAPS127A cells compared to control cells and YAPS94A cells. Secondly, we study the effects of YAP/TEAD interactions on the main biologic functions’ links to metastasis dissemination. We show an increase of cell migration using KHOS-S94A and KHOS-S127A, suggesting that YAP/TEAD interaction is not necessary to increase migration in OS. GSEA identifies genes related to epithelial-mesenchymal-transition (EMT), cell migration and TGF-β that are significantly overexpressed in YAPS127A and YAPS94A cells, strongly suggesting that TEAD is not required in YAP-driven expression of genes related to EMT, cell migration, and TGF-β signaling. Thirdly, using PLA and IP, we show an interaction of YAP/SMAD3 in OS cell line independently of YAP/TEAD interaction. In this context, using Tβ²R1 inhibitor, SD-208, we demonstrate the role of TGF-β/smad signaling in YAP-driven cell migration and metastatic process in OS. Finally, we investigated the effect of verteporfin, identified as an inhibitor of the Hippo signaling pathway, on OS progression. In vivo experiments showed that verteporfin reduces primary tumor growth and lung metastases development. In vitro experiments demonstrated that verteporfin decreases cell viability and inhibits the TGF-β/Smad3 cascade and the response of TGF-β key targets involved in the metastasis dissemination process. Together, these results defined the specific role of TEAD and SMAD3 in YAP-driven OS tumor and lung metastatic process. In addition, we demonstrated that YAP could be considered as a therapeutic target in OS. Citation Format: Sarah Morice, Geoffroy Danieau, Mathilde Mullard, Jérôme Amiaud, Régis Brion, Sarah Renault, Robel Tesfaye, Maryne Dupuy, Julie Talbot, Benjamin Ory, Bénédicte Brounais-Le Royer, Isabelle Corre, Anne Brouchet-Gomez, Françoise Rédini, Franck Verrecchia. Respective role of TEAD and Smad signaling in YAP-mediated osteosarcoma tumor growth and lung metastatic development [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A29.

Tumor Primary Location May Affect Metastasis Pattern for Patients with Stage IV NSCLC: A Population-Based Study.

Background Most patients with nonsmall cell lung cancer (NSCLC) were initially diagnosed with distant metastasis. At present, there is no study to clarify the correlation between the primary location of the tumor and the metastasis pattern in advanced NSCLC. So we conducted this study to explored the relationship between the tumor primary location and metastasis pattern in stage IV NSCLC. Methods A total of 19,295 eligible patients were identified from 2010 to 2012 in the SEER database. The main endpoint of our study was overall survival (OS). The survival curves were created by using the Kaplan–Meier method and compared by the usage of the Log Rank test. The clinical variable characteristics were compared by the chi-square test, and multivariate logistic regression analyses were used to evaluate the risk factors on metastasis patterns. All statistical P values were two-sided, and it was considered statistically significant when P ≤ 0.05. Results We found that different proportions of metastatic sites could be found in different tumor primary locations. In addition, the prognosis of lung metastasis was relatively good in patients with tumor location in main bronchus (P < 0.001), upper lobe (P < 0.001), lower lobe (P < 0.001) , and middle lobe (P = 0.005). Besides, there was no significant OS difference for patients whose primary location was overlapping lesion (P = 0.226). The results also demonstrated that compared with patients with primary tumor located in the main bronchus, those in the upper lobe were more likely to have brain metastasis (P = 0.01) and lung metastasis (P = 0.024), those in the middle lobe were more prone to develop lung metastasis (P = 0.035) and those in the lower lobe were more apt to cause bone metastasis (P = 0.005) and lung metastasis (P = 0.001). In addition, there was no statistical difference in metastasis patterns among patients with overlapping lesions (P > 0.05). Conclusions Different primary tumor locations might affect the metastasis pattern in patients with stage IV NSCLC.

Complete remission in a patient with advanced renal pelvis carcinoma with lung metastasis treated with durvalumab immunotherapy: a case report

For early-stage upper urothelial carcinoma, total nephroureterectomy combined with bladder sleeve resection is the standard treatment. However, for patients with advanced disease, there is a lack of effective therapeutic strategies. In recent years, with an increased understanding of cancer immunobiology, systemic immunotherapies targeting immune checkpoint inhibition has been explored and clinically used in the area of urothelial carcinoma. The programmed cell death 1 receptor (PD-1) and its ligand (PD-L1) are important negative regulators of immune activity, preventing the destruction of normal tissues and autoimmunity. Nowadays, five immune checkpoint inhibitors blocking PD-1 (pembrolizumab, nivolumab) or PD-L1 (atezolizumab, durvalumab, and avelumab) have been approved by the United States Food and Drug Administration (US FDA) for the first- or second-line use in urothelial carcinoma, based on durable response and manageable safety profiles observed in relevant clinical trials. In this study, we present the case of a 64-year-old patient with renal pelvis carcinoma who went on to develop lung metastasis after postoperative chemotherapy. CT scan showed multiple scattered solid small nodule foci in both lungs (considered as metastasis). The patient received immunotherapy with PD-L1 monoclonal antibody (Durvalumab) alone, and achieved complete remission (CR) after 3 cycles of treatment. During the treatment, slight weakness was reported, and no nausea, fever and other adverse events were observed. This case shows that durvalumab could effectively and safely treat a case of renal pelvis carcinoma with lung metastases.

Role of Platelet C-Type Lectin-Like Receptor 2 in Promoting Lung Metastasis in Osteosarcoma.

The overall prognosis of patients with sarcoma-based cancers has changed little in the last 20 years. There is an urgent need to investigate the metastatic potential of these tumors and to develop anti-metastatic drugs. It is becoming increasingly clear that platelets play an important role in the establishment of metastasis of carcinoma cells and could be a useful therapeutic target for patients with carcinoma. However, little is known about the role of platelets in sarcoma progression. Here, we investigated how osteosarcoma progression relates to platelet function to explore the possibility of anti-platelet therapy. We found that, similar to carcinoma cells, podoplanin (also known as Aggrus)-positive osteosarcoma cells induce platelet aggregation and activation. Administration of anti-glycoprotein Ibα (GPIbα, also known as CD42b) antibody reduced the lung metastasis of osteosarcoma. The supernatant from platelets cocultured with osteosarcoma cells contained several growth factors and promoted proliferation, invasiveness, and sphere formation of osteosarcoma cells in vitro. In addition, the development of lung metastasis was highly dependent on direct interaction between osteosarcoma cells and platelets. To explore the therapeutic target, we focused on the interactions between podoplanin on osteosarcoma and C-type lectin-like receptor (CLEC)-2 on platelets. The administration of a depleting antibody against CLEC-2 efficiently suppressed osteosarcoma metastasis into the lung. We also analyzed clinical data from patient samples at primary and metastatic sites. Although GPIbα expression was similar between the two sites, there was a significant increase in podoplanin at the metastatic site compared to that in the primary site, and the level of podoplanin expression in the primary site correlated with patient prognosis. These findings suggest that blockade of interactions between platelets CLEC-2 and osteosarcoma podoplanin represent the most promising therapeutic strategy for preventing the lung metastasis of osteosarcoma. © 2020 American Society for Bone and Mineral Research.

Prospective investigation of patent foramen ovale as a mechanism for brain metastasis in patients without prior lung involvement.

e14510 Background: Brain metastases can lead to significant morbidity and mortality in patients with advanced cancer. Preventing metastatic disease to the brain could thus substantially improve outcomes. The mechanism of brain metastasis is incompletely understood. Circulating tumor cells drain to the right heart and through the pulmonary circulation, where they may manifest as lung metastases, and can circulate further to the left heart and then to the brain. However, in patients who develop brain metastases without prior lung involvement, metastatic cells may take an alternate route. We hypothesized that cancer cells may pass directly from the right to left heart via a patent foramen ovale (PFO), akin to paradoxical embolism. The prevalence of PFO is approximately 20-30% in the general population; if further elevated in this population, PFO may play a role in brain metastasis development, and ultimately, prophylactic PFO closure may provide benefit. We conducted a pilot study to investigate whether PFO is associated with brain metastases in patients without prior lung involvement. Methods: We prospectively identified patients with brain metastases from a non-lung primary cancer with no preceding lung metastases. Participants underwent a transcranial Doppler study to assess for PFO. Agitated saline was injected intravenously at rest and with Valsalva maneuver. High intensity signals were counted in the middle cerebral arteries for 1 minute after each injection. Spencer grade ≥3 indicated a positive study, consistent with the presence of PFO. Results: We accrued 9 participants who met inclusion criteria. Primary cancers were breast (6 participants), upper gastrointestinal (2 participants), and thyroid (1 participant). A positive study was identified in 2/9 (22.2%) participants. One individual was a female with breast cancer who had no preceding extracranial metastases, and the other individual was a male with duodenal adenocarcinoma whose only prior metastatic disease was distant lymphadenopathy, not active at brain metastasis diagnosis. No participants have developed lung metastases as of their most recent imaging. Conclusions: In this prospective pilot study, we found no increased prevalence of PFO in patients who develop brain metastases without preceding lung involvement compared to estimates for the general population. Though a larger study is needed, the development of brain metastases in these patients may reflect tumors’ biological factors directing metastasis organotropism, rather than a structural pathway.

Identification of novel ligands that alter the function of type II NKT cells

Abstract Natural killer T (NKT) cells are distinct innate lymphocytes which recognize lipid antigens in the context of the major histocompatibility complex-like molecule CD1d. When activated, these cells can rapidly produce a variety of cytokines and modulate innate and adaptive immune responses. There are two main subsets of NKT cells, termed type I and type II NKT cells. Type I NKT cells are characterized primarily on the basis of their invariant TCRα expression and reactivity to the glycolipid α-galactosyl ceramide (αGalCer), whereas type II NKT cells express a different and more diverse TCR repertoire than type I NKT cells. Currently, the most widely studied antigen for type II NKT cells is sulfatide. Sulfatide-reactive type II NKT cells are reported to have the immunosuppressive function in autoimmune diseases and cancer. We previously showed a role for sulfatide-reactive type II NKT cells in suppressing tumor immunosurveillance in a murine model of lung metastasis. The activation of type II NKT cells in this model by the injection of sulfatide increased the development of lung metastasis. We synthesized diverse sulfatide-analogues varying in the number of double bonds and hydroxyl groups in the ceramide. In vitro, analogues having a phytosphingosine sphingoid base activated both type II and type I NKT cell hybridomas. On the other hand, the analogue C24:2 with a sphingosine base activated only the type II NKT cell hybridoma. In vivo, the C24:2 analogue significantly reduced the development of lung metastasis, showing distinct and opposite effects from sulfatide. The identification of new ligands inducing altered functional activity of type II NKT cells could be of great interest for developing new anti-tumor immunotherapies.

Abstract B84: Decipher the role of IL-33 as an activator of NK cells’ antitumor activity

Abstract Interleukin (IL)-33 is an alarmin belonging to the IL-1 family. Through its receptor ST2, IL-33 promotes both type 1 and type 2 immune responses, depending on the type of responding cells and the microenvironment in damaged tissues. Consistently, paradoxical roles of IL-33 have been reported in the context of cancer. These observations prompted us to investigate i) the expression of IL-33/ST2 in breast tumors and ii) the role of IL-33/ST2 in NK cell activation in physiologic and tumor contexts. Using immunohistochemistry (IHC) and transcriptomic analyses, we observed that IL-33 is expressed at higher levels in ductal carcinoma in situ (DCIS) compared to invasive breast cancer (IBC), especially in the stroma and in luminal BC subtype. In situ, IL-33 was mainly detected in endothelial cells and at lower extent in scattered cells within the stroma of breast tumors. Furthermore, we unraveled a new pathway for NK cells’ activation where IL-12 upregulates ST2 on human NK cells, which in turn become responsive to IL-33 by secreting high levels of IFN-γ and increasing their cytotoxic activity. This effect was specific to a subset of human CD56dim NK cells (20%) and was dependent on STAT-4 phosphorylation. In mice, we also observed a strong activation of spleen NK cell by IL-33 in combination with IL-12 in vitro. Finally, using IL-33ko mice, preliminary results show a contribution of endogenous IL-33 in the prevention of experimental lung metastasis development following B16 iv injection in therapeutic settings using exogenous administration of IL-12. Following up on these results, our aim is now to i) better characterize IL-33-responsive NK cells subpopulation in blood, ii) understand why human CD56bright NK cells do not upregulate ST2 despite response to IL-12 by phosphorylating STAT-4, iii) characterize NK cells’ response to IL-33 in human breast tumors, iv) evaluate ST2 expression by immune cells infiltrating breast and ovarian tumors, and v) confirm the activating role of IL-33 on NK cells’ biology in tumor models in vivo. All together, our observations are in favor of an NK-mediated antitumor role of IL-33 that we are currently pursuing as a potential novel therapeutic strategy in cancer. Citation Format: Anais Eberhardt, Elena Blanc, Emilie Lardenois, Sarah Renaudineau, Jennifer Herbulot, Benoit Dumont, Isabelle Durand, Emilie Charrier, Pauline Schmitt, Jean-Philippe Girard, Antoine Marçais, Thierry Walzer, Christophe Caux, Nathalie Bendriss-Vermare. Decipher the role of IL-33 as an activator of NK cells’ antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B84.