Development Of Pulmonary Metastases Research Articles

Abstract 6092: The dual roles of C-X-C MOTIF chemokine LIGAND 10 in pediatric osteosarcoma

Abstract Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of multi-agents chemotherapy and aggressive surgery, the survival of patients with osteosarcoma remains at 60% over the past three decades. In addition, the survival of patients who develop metastasis is dismal and often less than 25%. We have previously reported that serum C-X-C motif chemokine ligand 10 (CXCL10) is associated with survival in a large cohort of osteosarcoma patients. CXCL10 is an interferon-induced chemokine that binds to its cognate receptor CXCR3 to elicit its cellular effects via both autocrine and paracrine signaling. CXCL10 can promote tumor cell migration and metastasis via autocrine signaling, while it recruits CXCR3+ immune cells to exert a tumoricidal effect via paracrine signaling. However, little is known about the effect of circulating CXCL10 in cancer. To understand the role of CXCL10 in the pediatric bone tumor, we showed that a number of osteosarcoma cell lines expressed the CXCR3 protein. The addition of exogenous CXCL10 promoted tumor cell migration of three osteosarcoma cell lines but did not affect cell proliferation. However, the migratory effect of osteosarcoma cells is not specific to CXCL10, as other CXCR3-associated chemokines (CXCL4, CXCL9, and CXCL10) exerted similar effects. Using an orthotopic xenograft mouse model, we demonstrated intravenous injection of CXCL10 increased the development of pulmonary metastases of osteosarcoma. In contrast, expression profiling of osteosarcoma primary tumors showed that elevated expression of CXCL10 and CXCR3 was associated with a better prognosis, suggesting that a paracrine effect of CXCL10 by recruiting CXCR3+ immune cells to the tumor site. This notion is supported by our immune cell population deconvolution results, indicating that the predominant infiltrating immune cell types are macrophages and CD8+ T cells. Taken together, our results suggest a model in which CXCL10 may play both pro-tumor and anti-tumor roles in osteosarcoma. While a high level of exogenous or circulating CXCL10 may promote tumor cell migration and metastasis, the chemokine can also recruit CXCR3+ immune cells to exert a favorable prognostic effect. Further characterization of the sources of CXCL10 in the tumor site and circulation may lead to the development of a therapeutic approach that promotes CXCL10’s anti-tumor effects and minimizes its pro-tumor effects to improve the survival of osteosarcoma patients. Citation Format: Xiang Chen, Margaret Clement, John Hicks, Tsz Kwong Man. The dual roles of C-X-C MOTIF chemokine LIGAND 10 in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6092.

Abstract B26: Prometastatic effect of ICG-001, a β-catenin/CBP dependent transcription inhibitor, in osteosarcoma

Abstract Osteosarcoma is the most common malignant bone tumor in the pediatric population, representing around 4 cases per million people per year in the world. Patient survival is closely related to the response of tumor cells to chemotherapy, reaching 70% at 5 years for patients with localized disease, but only 25% for high-risk patients with relapsed or metastatic disease. In 20% of cases, patients have detectable metastases at diagnosis, preferentially in the lungs, which is a sign of poor prognosis. However, it is estimated that around 80% of patients have pulmonary micrometastases, sometimes undetectable at diagnosis by imaging techniques. This makes metastatic dissemination a significant issue in the management of osteosarcoma. In addition, the survival rate has not evolved since the past decades and there is no current efficient therapy for these patients. Therefore, it is necessary to develop new therapeutic strategies. In this context, a dysregulation of the canonical Wnt signaling pathway (Wnt/β-catenin) has been reported in many osteosarcoma cases, but its implication in the development of primary and metastatic osteosarcoma is still controversial. An increase in β-catenin expression has been described in human osteosarcoma tissues compared to normal bones. Thus, we evaluated the antitumor potential of ICG-001, a small molecule that specifically binds to the transcriptional co-activator CREB-Binding Protein (CBP), disrupting its interaction with β-catenin and thus suppressing the Wnt/β-catenin target gene expression. First, we demonstrated that ICG-001 inhibits β-catenin/CBP-dependent transcription as evaluated by TCF-LEF reporter assay and RT-qPCR on β-catenin target genes in three human osteosarcoma cell lines, KHOS, MG63, and SJSA1. Moreover, ICG-001 decreases proliferation of osteosarcoma cells in a dose- and time-dependent manner, associated with a cell cycle blockade in G0/G1 phase after 24h of treatment, but without any effect on cell death. However, surprisingly, ICG-001 promotes KHOS, MG63, and SJSA1 cell migration in vitro and the development of pulmonary metastases in a murine xenograft model of osteosarcoma induced by injection of KHOS cells in a paratibial site. This study therefore raises new questions about the role of the Wnt/β-catenin pathway in the metastatic development of osteosarcoma. Thus, it is necessary to identify the different signatures of the signaling pathways potentially activated after disruption of the β-catenin and CBP interaction in osteosarcoma cells and responsible for the prometastatic effect of this drug. Citation Format: Geoffroy Danieau, Sarah Morice, Sarah Renault, Kevin Biteau, Régis Brion, Jérôme Amiaud, Frédéric Lezot, Franck Verrecchia, Françoise Rédini, Bénédicte Brounais-Le Royer. Prometastatic effect of ICG-001, a β-catenin/CBP dependent transcription inhibitor, in osteosarcoma [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 B26.

Abstract PR13: Surgical excision of the primary tumor in osteosarcoma model results in enhanced metastatic growth by modulating the lung immune microenvironment

Abstract Surgery has been shown to enhance metastasis in a number of cancers. To examine this in the context of osteosarcoma, we implanted syngeneic murine K7M2 cells into the tibia of BALB/c mice, which we had shown previously to result in immediate seeding of K7M2 cells into the lung. One week post-implantation, mice were assigned to either control or surgical groups and all mice were euthanized at week 4 post-implantation. Mice that underwent amputation of the primary tumor displayed an increase in the number of pulmonary surface nodules as compared to tumor-bearing control animals (17 vs. 9.5; p<0.05), showing that excision of the primary tumor indeed enhances metastasis in this model. As we previously showed that K7M2 metastasis without surgical intervention is strongly inhibited by re-educating tumor-associated macrophages (TAMs) with gefitinib, we also examined the effect of gefitinib on surgery-accelerated metastasis. We found that perioperative gefitinib abrogated the effects of primary tumor removal on the development of pulmonary metastases (17 vs. 6; p< 0.01), strongly suggesting that TAMs also mediate surgery-accelerated metastasis. To examine changes in the pulmonary immune milieu, we utilized the same model of surgery-accelerated metastasis. Lungs were homogenized and immune cell populations were analyzed using flow cytometric analysis at 48 hours and 3 weeks after surgery. We found that surgery caused a significant reduction in antitumor macrophages, identified by MHCII positivity (21.1% vs. 13.2%; p<0.01), and a significant increase in protumor macrophages, identified by CD206 positivity (33.0% vs. 42.4%; p<0.01), at 48 hours after surgical resection. There was no change in the proportion of CD4+ or CD8+ T cells at this time point. At 3 weeks post-surgery the proportion of macrophage populations became similar for both groups, but there was now a small but significant reduction in the CD4+ and CD8+ T cells in the surgical group (68.4% vs. 60.3%; p<0.05 and 25.0% vs. 19.3%; p<0.01). In conclusion, surgical removal of the primary tumor accelerated the growth of pulmonary metastases in a murine model of OS. Gefitinib was able to abrogate this effect, suggesting macrophages as key cellular mediators. Furthermore, surgery altered the macrophage phenotype within the lungs in the acute postoperative period toward a protumor state, subsequently reducing the CD4+ or CD8+ T-cell populations. Taken together these data suggest that surgery contributes to enhanced metastatic outgrowth by altering the immune microenvironment to an immunosuppressive and protumor state. This abstract is also being presented as Poster B38. Citation Format: Michelle P. Kallis, Caroline Maloney, Morris Edelman, Samuel Z. Soffer, Marc Symons, Bettie M. Steinberg. Surgical excision of the primary tumor in osteosarcoma model results in enhanced metastatic growth by modulating the lung immune microenvironment [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 PR13.

TH1579, MTH1 inhibitor, delays tumour growth and inhibits metastases development in osteosarcoma model.

BackgroundOsteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers.MethodsThe aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases.FindingsMTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90 mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases.InterpretationAll these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma.FundingThis study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.

Mislocalized cytoplasmic p27 activates PAK1-mediated metastasis and is a prognostic factor in osteosarcoma.

The development of pulmonary metastasis is the leading cause of death in osteosarcoma (OS), which is the most common malignant bone tumor in children. We have previously reported that the tumor suppressor p27 (KIP1, CDKN1B) is frequently mislocalized to the cytoplasm of OS. However, its prognostic significance and metastatic mechanism are still elusive. Here, we show that cytoplasmic p27 significantly correlated with a higher metastatic status and poorer survival of OS patients (n = 136, P < 0.05), highlighting the clinical significance of p27 mislocalization in OS. Mechanistically, cytoplasmic p27 is co‐immunoprecipitated with p21‐activated kinase 1 (PAK1), which resulted in higher PAK1 phosphorylations, actin polymerization, and cell motility in p27‐mislocalized OS cells. Silencing PAK1 expression in different p27‐mislocalized OS cell lines decreased the migratory and adhesion abilities in vitro, as well as the development of pulmonary metastases in vivo. Similar PAK1‐dependent motility was also observed in other p27‐mislocalized cancer cell lines. In summary, our study suggests that cytoplasmic p27‐mediated PAK1 activation is crucial for OS metastasis. A biomarker‐guided targeted therapeutic approach for metastatic OS and other cancers harboring p27 mislocalization can be developed, where cytoplasmic p27 is used for risk stratification and PAK1 can be exploited as a potential therapeutic target.

Abstract 2876: p21-activated kinase 1 is a novel therapeutic target for metastatic osteosarcoma harboring p27 mislocalization

Abstract Lung metastasis is the leading cause of death in osteosarcoma (OS), the most common bone cancer in children and young adults. Due to a lack of biomarkers and therapeutic targets, significant progress has not been made in the treatment of metastatic OS over the past three decades. The survival of metastatic patients remains dismal. We have previously reported that the tumor suppressor protein p27 (KIP1, CDKN1B) is frequently mislocalized in the cytoplasm of OS, which promotes the motility and invasiveness of the tumor cells and the development of pulmonary metastases in mice. In this study, we showed that a higher proportion score of cytoplasmic p27 significantly correlated with metastases developed within 3- or 5-year of follow-up, and poor disease-specific and event-free survival in a large OS cohort (n=136). Using immunoprecipitation followed by mass spectrometry, we demonstrated that cytoplasmic p27 interacted with p21-activated kinase 1 (PAK1), where the two proteins co-localized in the cytoplasm of OS cells. PAK1 is a serine/threonine kinase that has been implicated in various cancers; however, its role in OS is still elusive. We showed that the phosphorylations at serine-144 and threonine-423 residues of PAK1 were higher in the p27-mislocalized cells relative to the control cells. The PAK1 phosphorylations increased actin stress fiber formation in the tumor cells. Gene silencing or therapeutic inhibition of PAK1 reduced the motility of p27-mislocalized cells in vitro. Similar results were observed in other cancer cell lines known to have the p27 mislocalization, suggesting PAK1 is a common and potential downstream effector of p27-mediated metastasis. An experimental metastasis mouse model further showed that a PAK1-silenced mutant had a significant impairment in the development of pulmonary metastasis. In summary, our study indicates that cytoplasmic p27 is a novel prognostic biomarker in OS. In the tumor cell, the mislocalization activates PAK1-mediated cell motility and metastasis. Targeting PAK1 in metastatic OS harboring the p27 mislocalization may be exploited as a potential therapeutic intervention to improve the poor outcome of the affected children. Citation Format: Xiang Chen, Justin Cates, Yuchen Du, Yun Jung Sung, Xiao-nan Lo, John Hicks, Tsz-Kwong Man. p21-activated kinase 1 is a novel therapeutic target for metastatic osteosarcoma harboring p27 mislocalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2876.

Abstract 3210: A potential immunotherapeutic approach for the treatment of osteosarcoma

Abstract Purpose: Survival of osteosarcoma (OS) patients has remained stagnant for the past 30 years thus the need for new therapeutic strategies. NKTR-214 is a novel cytokine therapy that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rbeta gamma) so that it can selectively expand and proliferate CD8+ T cells. Compared to recombinant human IL-2, NKTR-214 demonstrated superior therapeutic benefit in a melanoma mouse model and was well-tolerated. Because systemic IL-2 has shown limited efficacy against OS, we hypothesize that NKTR-214 could potentially offer increased therapeutic benefit. Methods: The K7M2/K7M3 disseminated and orthotopic OS mouse models were used. Three doses of NKTR-214 were given intravenously every 9 days. In the K7M2 disseminated model a prophylactic or therapeutic approach was taken where treatment was started right after tumor injection or when there was evidence of lung metastases. In the K7M3 orthotopic model, treatment was started once tumor was established (day 20) or 10 days after tumor injection. Bone tumor size was assessed by serial plain X-rays. Immunohistochemistry was performed to assess histologic tumor response (bone and lung) as well as evidence of immune cell infiltrate. Tumor immune infiltrate was analyzed by flow cytometry. Results: We demonstrated therapeutic effect of NKTR-214 as a single agent. In the disseminated model, NKTR-214 administered every 9 days i.v. prophylactically resulted in an increase in the median survival of mice with OS lung metastases (21 days untreated vs. 51 days NKTR-214). Survival was similar when therapeutic treatment was started after tumor nodules were apparent in the lung and continued every 9 days (51 days vs 52.5 days). In the orthotopic model, we demonstrated NKTR-214 efficacy against K7M3 primary bone tumor and the development of pulmonary metastases. The total tibia tumor area in the NKTR-214-treated group was significantly lower than in the untreated group (p&amp;lt;0.01). Likewise, the total lung metastases tumor area as well as the number of micrometastases was significantly lower in the treated as compared to the untreated group (p&amp;lt;0.009). We also demonstrated increase in the number of CD8+ and CD4+ T cells in mice treated with NKTR-214 single agent as compared to the untreated group. This data provides rationale for the use of NKTR-214 alone or in combination with checkpoint inhibitors or adoptively transferred NK cells or T cells as a potential effective therapy for OS. Conclusion: In conclusion, our findings demonstrate that NKTR-214 is effective in controlling the growth of primary OS, regrowth of tumor after amputation and inhibition of lung metastasis formation. These pre-clinical studies provide the basis for potential translation of NKTR-214-based immunotherapeutic regimens for OS into the clinic. Citation Format: Marlene Hennessy, Souri Felix, Andrew Wahba, Saul Kivimae, Takahiko Miyama, Mariella Cabrera, Maria Gabriela Segura, Jonathan Zalevsky, Willem Overwijk, Nancy Gordon. A potential immunotherapeutic approach for the treatment of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3210.

Surgical outcomes of pulmonary metastasis from hepatopancreatobiliary carcinomas: a comparison with pulmonary metastasis from colorectal carcinomas.

Surgical indications for pulmonary metastasis from hepatopancreatobiliary (HPB) carcinomas remain controversial. Between 2000 and 2015, 25 patients with pulmonary metastasis from HPB carcinomas and 145 with that from colorectal carcinomas underwent metastasectomies in our institution. The primary diseases were hepatocellular carcinoma (HCC) in 8 patients, pancreatic carcinoma (PC) in 12 and biliary tract carcinoma (BTC) in 5. All patients had a sufficient pulmonary reserve, controlled primary disease and no evidence of other metastatic disease. Perioperative factors were investigated retrospectively to analyze the overall survival (OS), pulmonary metastasis-free survival (PmFS) after pulmonary metastasectomy and disease-free interval between surgery for primary disease and the development of pulmonary metastasis (DFI). Complete resection was performed in all patients with lobectomy in 3, segmentectomy in 5 and partial resection in 17. The respective 1-, 2- and 5-year OS rates after metastasectomy were 82.6%, 69.8% and 69.8% in HPB patients and 98.3%, 92.4% and 78.0% in colorectal carcinoma patients (p = 0.351). The 2-year PmFS of HPB patients was 80.0%, versus 60.6% for colorectal carcinoma patients (p = 0.265). The DFI was 41.4months for HPB patients and 34.5months for colorectal carcinoma patients (p = 0.273). Metastasectomy for pulmonary metastasis from HPB may be performed in carefully selected patients.

A Virus-Like-Particle immunotherapy targeting Epitope-Specific anti-xCT expressed on cancer stem cell inhibits the progression of metastatic cancer in vivo

ABSTRACTAggressive forms of breast cancer, such as Her2+ and triple negative breast cancer (TNBC), are enriched in breast cancer stem cells (BCSC) and have limited therapeutic options. BCSC represent a key cellular reservoir for relapse, metastatic progression and therapeutic resistance. Their ability to resist common cytotoxic therapies relies on different mechanisms, including improved detoxification. The cystine-glutamate antiporter protein xCT (SLC7A11) regulates cystine intake, conversion to cysteine and subsequent glutathione synthesis, protecting cells against oxidative and chemical insults. Our previous work showed that xCT is highly expressed in tumorspheres derived from breast cancer cell lines and downregulation of xCT altered BCSC function in vitro and inhibited pulmonary metastases in vivo. We further strengthened these observations by developing a virus-like-particle (VLP; AX09-0M6) immunotherapy targeting the xCT protein. AX09-0M6 elicited a strong antibody response against xCT including high levels of IgG2a antibody. IgG isolated from AX09-0M6 treated mice bound to tumorspheres, inhibited xCT function as assessed by reactive oxygen species generation and decreased BCSC growth and self-renewal. To assess if AX09-0M6 impacts BCSC in vivo seeding, Her2+ TUBO-derived tumorspheres were injected into the tail vein of AX09-0M6 or control treated female BALB/c mice. AX09-0M6 significantly inhibited formation of pulmonary nodules. To evaluate its ability to impact metastases, AX09-0M6 was administered to mice with established subcutaneous 4T1 tumors. AX09-0M6 administration significantly hampered tumor growth and development of pulmonary metastases. These data show that a VLP-based immunization approach inhibits xCT activity, impacts BCSC biology and significantly reduces metastatic progression in preclinical models.

Higher local recurrence rates after intralesional surgery for giant cell tumor of the proximal femur compared to other sites.

The treatment of giant cell tumor (GCT) of bone remains controversial. Intralesional surgery (curettage) results in a higher rate of local recurrence, but better functional results compared to resection. The aim of this study was to assess whether the use of curettage was successful in the treatment of GCT of long bones. We evaluated the influence of adjuvant treatment, local tumor presentation, and demographic factors on the risk of recurrence. We retrospectively reviewed the records of patients treated for GCT of long bones between 1990 and 2013, using curettage. No patient had any treatment other than surgery. After detailed curettage, the bone cavity was filled with bone allografts and/or cement. Recurrence rates, risk factors for recurrence and the development of pulmonary metastases were determined. The minimum follow-up was 24months. We enrolled 210 patients with GCT of long bones treated by curettage. The rate of local recurrence was 16.2% (34/210 patients). The median follow-up was 89.2months. In the multivariate analysis, no significant statistical effect on the local recurrence rate could be identified for gender, patient's age, Campanacci's grading, or cement versus bone allografts. The only independent risk factor related to the local recurrence was the site, with a statistically significant higher risk for patients with GCT of the proximal femur. Our observation on the correlation of tumor location and risk of local recurrence is new. We suggest that patients with GCT of bone in the proximal femur should be followed closely soon after surgery to identify any possible recurrence.

Abstract B46: miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas

Abstract The purpose of this study is to better delineate the implication of the TAp73β/miRNA-193a-5p's axis in the Cisplatine chemoresistance, in the context of Osteosarcoma and Ewing Sarcoma, the two most common Bone Sarcomas. The last decade's improvement in the biomedical research field has widely contributed to improve the Bone Sarcomas outcome. Among the arsenal of chemotherapeutic-drugs currently used in those pathologies, the Cisplatine is a very common therapeutic option. Unfortunately, chemoresistance against this drug remains, often leading to the development of pulmonary metastases and the consequent relapse of the young patients suffering from these tumors. In the hope of eradicating these aggressive kinds of cancer, a better understanding of the molecular origins of such chemoresistance is needed. Through their gene's expression inhibitory role, the implication of the miRNAs in the Bone Sarcomas' chemoresistance is already clearly established. Furthermore, it has previously been demonstrated that through targeting the p53-family member TAp73β, the miR-193a-5p is implicated in the Cisplatine resistance in a carcinoma model. Thus, the aim of this work is to assess if TAp73β is also a mediator of the Cisplatine-induced apoptosis in Bone Sarcomas and consequently, if its modulation by the miR-193a-5p could be implicated in the chemoresistance mechanisms against this drug. In addition, this study also strives to bring the proof of concept of miR-193a-5p's as a new therapeutic-target to potentiate the Cisplatine anti-tumor's effects in Bone Sarcomas and override chemoresistance. Our qRT-PCR and viability assays results reveal that human Osteosarcoma and Ewing Sarcoma cell lines express the miR-193a-5p and TAp73β at various levels and display different Cisplatine sensitivities. In addition, an interesting correlation was found between the Cisplatine IC50 values and the miR-193a-5p expression levels. In line with these results, we have also demonstrated for the first time in this model that Cisplatine represses the miR-193a-5p expression and consequently induces TAp73β and its target-genes. Through TAp73β expression level modulation, we validated its implication in both the apoptosis and the clonogenic capabilities of the Bone Sarcoma cells. Our cellular viability assays also confirm the predicted role of TAp73β-mediated Cisplatine-sensitivity. In the same way, we show that modulating the miR-193a-5p expression level through transient transfections with synthesis miRNAs (pre-miR) or their inhibitory counterparts (anti-miRs) has a functional impact on the Cisplatine sensitivity and the apoptosis-induction (caspase 3/7 activity assay, PARPs cleavage). In conclusion, our body of data confirms that through targeting TAp73β, the miRNA-193a-5p modulates the viability, the clonogenic capabilities and the Cisplatine-induced apoptosis in Bone Sarcoma cells. Such results shed light on the implication of the miR-193a-5p in the Bone Sarcomas' Cisplatine-chemoresistance and open the road to new therapeutic opportunities provided by targeting the miR-193a-5p/TAp73β axis in the context of these pediatric cancers. In a fundamental approach, studying the Cisplatine-resistance might lead to better understand the role of miRNAs in therapeutic resistance. However, this miRNAs could also be used as “chemo-sensitizer” molecule in this context, improving the Bone Sarcoma outcome. Citation Format: Camille Jacques, François Lamoureux, Marc Baud'huin, Lidia Rodriguez Calleja, Dominique Heymann, Benjamin Ory. miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B46.

Abstract LB-113: Recombinant HER2/Neu expressing Listeria combined with radiation safely delays tumor progression and prolongs overall survival in a phase I clinical study in canine osteosarcoma

Abstract The purpose of this study is to determine the safety and efficacy of a highly attenuated recombinant Listeria monocytogenes expressing a chimeric human HER2/neu (Lm-LLO-HER2/neu) in combination with palliative radiation (RT) to induce HER2 specific immunity and delay tumor progression in dogs with spontaneous osteosarcoma (OSA). Dogs develop OSA that recapitulates many aspects of human OSA, and are recognized as a clinically relevant, large animal model in which to evaluate novel therapies. Previously we have shown that Lm-LLO-HER2/neu administration to dogs with HER2+ OSA following amputation and chemotherapy is safe, breaks tolerance to HER2 and prolongs overall survival. Ten systemically healthy dogs with histopathologically confirmed, treatment naïve, HER2+ appendicular OSA, and no evidence of cardiac or metastatic disease were enrolled. All dogs received 2 × 8Gy fractions of RT on consecutive days, followed by the first of 8 intravenous doses of Lm-LLO-HER2/neu given once every 3 weeks. Dogs were monitored at each treatment and every 2 months after for systemic and cardiac toxicity, lameness and quality of life (QOL) (using a validated owner questionnaire). Radiographs were performed at baseline, week 10, week 22 and every 2 months thereafter to determine the effect on the primary tumor and development of pulmonary metastases. PBMCs were collected every 3 weeks to evaluate HER2/neu specific T cell responses. The primary endpoint was time to progression; secondary end points were safety and overall survival. Results are compared to historical canine controls that received 2 × 8Gy RT alone. Repeat Lm-LLO-HER2/neu administration was well tolerated with no systemic or cardiac toxicity. Lameness and QOL showed near uniform improvement over the study period. To date, limb radiographs of 7 dogs have been taken 10 weeks post enrollment and showed no evidence of tumor progression. 4 dogs have currently completed the scheduled 8 vaccinations, and limb radiographs of these dogs taken at 22 weeks, showed no progression of the primary lesion. At the time of writing, 4/10 dogs have died; 2 dogs were euthanized due to pathological fracture, and 2 dogs were euthanized due to metastatic disease. One dog, still alive, has developed pulmonary metastatic disease. At the time of writing, median time to progression is 243 days and median survival time (MST) is 285 days. Historically, MST of dogs treated with RT alone is 136 days. Results of IFN-γ ELISpot assays are pending. To conclude, our preliminary results show repeat Lm-LLO-HER2/neu administration is safe and well tolerated with no cardiac toxicity. Our results also suggest that combination RT and Lm-LLO-HER2/neu immunotherapy improves QOL, delays primary tumor progression and prolongs overall survival in canine OSA. These findings may have important translational relevance for human patients with OSA and other HER2/neu+ cancers. Citation Format: Nicola Mason, Josephine Gnanandarajah, Danielle Laughlin, Julie Engiles, Anu Wallecha, Yvonne Paterson. Recombinant HER2/Neu expressing Listeria combined with radiation safely delays tumor progression and prolongs overall survival in a phase I clinical study in canine osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2015-LB-113

Abstract 1530: Therapeutic efficacy of cancer stem cell vaccine in an adjuvant setting

Abstract The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. Novel therapeutic strategies are needed to immunologically target CSCs. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DC). Using mouse models we demonstrate that DC pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (CSC-DC) significantly inhibited local tumor growth, reduced development of pulmonary metastases and prolonged survival in the minimal tumor models. The effect was associated with down regulation of CCR7 and CCR10 in tumor cells and decreased CCL27 and CCL28 expression in lung tissue. CSC-DC vaccine significantly reduced ALDHhigh CSCs. Direct targeting of CSCs was demonstrated by specific binding of IgG produced by ALDHhigh CSC-DC vaccine-primed B cells to ALDHhigh CSCs, resulting in lysis of these target cells in the presence of complement. When administered in the adjuvant setting after surgical excision of the bulk tumor mass, administration of CSC-DC vaccine reduced development of local tumor growth as well as systemic disease and prolonged survival demonstrating the efficacy of this approach. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after surgery. Note: This abstract was not presented at the meeting. Citation Format: Yangyang Hu, Lin Lu, Alfred E. Chang, Martin Egenti, John Owen, Jeffrey S. Moyer, Mark E.P. Prince, Jianchuan Xia, Shiang Huang, Max S. Wicha, Qiao Li. Therapeutic efficacy of cancer stem cell vaccine in an adjuvant setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2015-1530

Abstract 1410: Evaluation of the immunogenicity of ALDHhigh human head and neck squamous cell carcinoma cancer stem cells in vitro

Abstract Using mouse models we reported that dendritic cells (DC) pulsed with cancer stem cells (CSC) enriched by virtue of their expression of the CSC marker aldehyde dehydrogenase (ALDH) significantly reduced development of pulmonary metastases and prolonged survival. In this recent study, we established the concept that the antigenicity/immunogenicity of ALDHhigh human head and neck squamous cell carcinoma (HNSCC) cancer stem cells is distinct from that of ALDHlow non-CSCs. To this end, we generated CSC-loaded DCs to sensitize autologous peripheral blood lymphocytes to react with CSCs using human HNSCC samples in vitro. From peripheral blood and paired tumor tissues collected from 9 patients with HNSCC, we obtained PBMCs and established 4 HNSCC tumor cell lines. DCs generated from the PBMC and pulsed with the lysate of ALDHhigh cells isolated from cultured HNSCC cells (CSC-DC) could sensitize autologous T, B lymphocytes in vitro, which was evident by cytokine production, CTL activity, and antibody secretion of these primed T, B cells in response to ALDHhigh CSCs. In contrast, DCs pulsed with lysate of ALDHlow cells from the same HNSCC patient (ALDHlow-DC) resulted in limited sensitization/priming of autologous T, B lymphocytes to produce IFNγ, lyse CSCs, and secrete IgM and IgG in response to ALDHhigh CSCs. These results demonstrated significant differences in the antigenicity/immunogenicity between ALDHhigh CSCs vs. ALDHlow cells isolated from the tumor specimen of patients with HNSCC, which indicates the existence of unique CSC antigens in the ALDHhigh population. In addition, this study demonstrates that it is feasible to generate DCs from the PBMCs and isolate ALDHhigh CSCs from cultured tumor cells of the patients with HNSCC to prepare CSC-DC vaccines for clinical application. Citation Format: Mark E.P. Prince, Li Zhou, Yang Xia, Huimin Tao, Jeffrey S. Moyer, Lin Lu, John Owen, Alfred E. Chang, Jianchuan Xia, Gregory Wolf, Max S. Wicha, Xiubao Ren, Yangyi Bao, Shiang Huang, Qiao Li. Evaluation of the immunogenicity of ALDHhigh human head and neck squamous cell carcinoma cancer stem cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1410. doi:10.1158/1538-7445.AM2015-1410

The immunotherapy of canine osteosarcoma: a historical and systematic review.

Osteosarcoma is a malignant mesenchymal neoplasm that accounts for the majority of primary bone tumors in dogs and shares biological and clinical similarities with osteosarcoma in humans. Despite dose intensification with conventional cytotoxic therapies, survival times for dogs and humans diagnosed with high‐grade osteosarcoma have not changed in the past 20 years, with the principal cause of mortality being the development of pulmonary metastases. Given the therapeutic plateau reached for delaying metastatic progression with cytotoxic agents, exploration of alterative adjuvant therapies for improving management of osteosarcoma micrometastases is clinically justified. Evidence suggests that osteosarcoma is an immunogenic tumor, and development of immunotherapies for the treatment of microscopic lung metastases might improve long‐term outcomes. In this review, the history and foundational knowledge of immune interactions to canine osteosarcoma are highlighted. In parallel, immunotherapeutic strategies that have been explored for the treatment of canine osteosarcoma are summarized. With a greater understanding and awareness for how the immune system might be redirected toward combating osteosarcoma metastases, the rational development of diverse immune strategies for managing osteosarcoma holds substantial promise for transforming the therapeutic landscape and improving disease management in both dogs and human beings.

Predicting pulmonary metastases among patients with indeterminate pulmonary nodules with renal cell carcinoma.

505 Background: Indeterminate pulmonary nodules (IPN) are of uncertain significance in renal cell carcinoma (RCC) patients. We sought to determine the natural history of IPN in patients undergoing radical nephrectomy and to identify clinical variables associated with the development of lung metastases. Methods: We reviewed all radical nephrectomy patients at a single institution from 2005 – 2009 who had ≥1 IPN on chest computed tomography (CT) within 6 months prior to surgery and no evidence of distant metastates. All chest CTs were re-reviewed by a radiologist who was blinded to outcomes to independently determine number, size, location of nodules and whether the IPN appeared concerning for metastases based on nodule size, tissue density, and morphology. Univariate and multivariate Cox regression was used to assess associated predictive factors. Results: Of 258 patients with IPN, 73 (28%) developed pulmonary metastases. Median follow−up was similar in the groups at 36 months. Patients who developed pulmonary metastases had significantly poorer survival compared to those who did not (3-year DSS 54.5 versus 86.5 months, p&lt;0.001), and 39 (53%) developed pulmonary metastases at the same location as the largest IPN. On univariate analysis, larger renal tumor diameter (HR 1.12, p&lt;0.001), number of pulmonary nodules CT (HR 1.12, p 0.001), presence of hilar lymphadenopathy on chest CT were associated with both lung metastasis development and cancer-specific mortality. After adjusting for age, performance status, comorbidities, histology, and smoking status, higher tumor grade (HR 5.11, p 0.007), presence of tumor thrombus above the diaphragm (HR 4.38, p 0.013), perinephric (HR 4.29, p 0.002) or renal sinus (HR 2.16, p 0.045) fat invasion, and a chest CT concerning for metastasis by the radiologist (HR 2.91, p&lt;0.001) were associated with development of pulmonary metastases. Conclusions: RCC patients with IPN who develop pulmonary metastases have a significantly poorer outcome. Independent predictors of developing lung metastases include tumor grade, presence of tumor thrombus above the diaphragm, perinephric or renal sinus fat invasion, and a chest CT concerning for metastasis.

Abstract B07: Using magnetic resonance and hyperpolarized tracers to monitor solid tumor progression, metastasis, and response to treatment

Abstract Purpose: To optimize translational fidelity of a murine model of anaplastic thyroid carcinoma (ATC) using real-time metabolic imaging. Background: Anaplastic thyroid carcinoma (ATC) is an aggressive solid malignancy with rapid growth, invasion of critical cervical structures and a high rate of pulmonary metastasis. Although rare in overall incidence (2-3% of thyroid cancer), ATC accounts for 50% of thyroid cancer related mortality. The rapid rate of growth and metastasis makes optimization of treatment algorithms challenging and development of novel therapeutic strategies difficult. Our group has developed an integrated preclinical model of ATC using validated human cell lines and a xenograft mouse model which recapitulates disease characteristics and closely mimics response to treatment including chemotherapy and ionizing radiation (IR). Experimental procedures: Validated human ATC cell lines were either injected into the right thyroid lobe of athymic nude mice in an orthotopic tumor model or intravenously (tail-vein) to generate pulmonary metastases. Tumors were exposed to varying doses of ionizing radiation (IR) and monitored using both anatomic magnetic resonance imaging (MRI) sequences as well as hyperpolarized (HP) [1-13C]-pyruvate (PYR). HP-MR data was acquired using either temporal or spatial acquisition sequences depending on the experimental aims. At the completion of intended experiments, primary thyroid tumors and pulmonary metastases were harvested and analyzed using standard histologic and immunohistochemical techniques. Results: We have employed HP-MR using labeled [1-13C]-pyruvate to measure tumor metabolic activity in real-time. We have translated this capability into precise measurements of tumor viability at the primary site, which when combined with anatomic MRI sequences provides enhanced visualization and identification of critical cervical structure invasion. HP-MR has allowed us to identify sub-centimeter pulmonary metastases and differentiate tumor from normal lung parenchyma based on the inherently higher rate of conversion of labeled pyruvate (PYR) into lactate (LAC). We had previously demonstrated that pharmacologic perturbations in tumor reducing potential can be detected in real time using HP-MR. In these studies we extended these initial experiments to demonstrate that HP-MR can detect parallel and opposing changes in tumor reactive oxygen species (ROS) levels following irradiation. Using the ATC xenograft mouse model we were able to measure acute (2hr) changes in tumor reducing potential following irradiation compared to control tumor (p=0.004). Furthermore, HP-MR allowed us to identify areas of ATC tumors which display enhanced reducing potential, potentially driving ATC radio-resistance. Conclusion: Utilization of HP-MR metabolic imaging has allowed us to further develop our preclinical model of ATC and improve our ability to develop novel translational applications. Specifically, we believe HP-MR can improve the ability to monitor loco-regional tumor growth and invasion, as well as the development of pulmonary metastases. Furthermore, the development of a quantifiable imaging-based biomarker of radiation effect may permit adaptive IR plans based upon tumor response that would optimize disease treatment and improve clinical outcomes. Citation Format: Vlad Sandulache, Yunyun Chen, Jaehyuk Lee, Marc Ramirez, Heath Skinner, Laurence Court, Jim Bankson, Stephen Lai. Using magnetic resonance and hyperpolarized tracers to monitor solid tumor progression, metastasis, and response to treatment. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B07.

Abstract 4796: Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells

Abstract The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DC). Using mouse models we demonstrate that DC pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (CSC-DC) significantly reduced development of pulmonary metastases and prolonged survival. The effect was associated with down regulation of CCR7 and CCR10 in tumor cells and decreased CCL27 and CCL28 expression in lung tissue. CSC-DC vaccine significantly reduced ALDHhigh CSCs in the primary tumors. Direct targeting of CSCs was demonstrated by specific binding of IgG produced by ALDHhigh CSC-DC vaccine-primed B cells to ALDHhigh CSCs, resulting in lysis of these target cells in the presence of complement. When administered in the adjuvant setting after localized radiation therapy of established tumors, CSC-DC vaccine reduced development of local tumor growth as well as systemic disease and prolonged survival demonstrating the efficacy of this approach. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after conventional treatment of cancers. Citation Format: Lin Lu, Huimin Tao, Yang Xia, John Owen, Jeffrey S. Moyer, Mark E.P Prince, Alfred E. Chang, Max S. Wicha, Qiao Li. Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4796. doi:10.1158/1538-7445.AM2014-4796

Mesenchymal Stromal Cell Dependent Regression of Pulmonary Metastasis from Ewing's.

Introduction: Ewing’s sarcoma (ES) is the second most common bone tumor in children. Survival has not improved over the last decade and once pulmonary metastatic disease is present, survival is dismal. Mesenchymal stromal cell (MSC) therapy has shown potential benefit for Kaposi’s sarcoma; however, the role of progenitor cell therapies for cancer remains controversial. MSC treatment of ES or pulmonary metastatic disease has not been demonstrated. We have developed an orthotopic xenograft model of ES in which animals develop spontaneous pulmonary metastases. Within this model, we demonstrate the use of MSCs to target ES lung metastasis.Materials and Methods: Human ES cells were transfected with luciferase and injected into the rib of nude mice. Development of pulmonary metastases was confirmed by imaging. After flow cytometry based characterization, MSCs were injected into the tail vein of nude mice with established local ES tumor or pulmonary metastasis. Mice were treated with intravenous MSCs weekly followed by bioluminescent imaging.Results: The intravenous injection of MSCs in an ES model decreases the volume of pulmonary metastatic lesions; however, no effect on primary chest wall tumor size is observed. Thus verifying the MSC preferential homing to the lung. MSCs are found to “home to” the pulmonary parenchyma and remain engrafted up to 5 days after delivery.Discussion: MSC treatment of ES slows growth of pulmonary metastasis. MSCs have more affinity for pulmonary metastasis and can effect a greater decrease in tumor growth in the lungs compared to the primary tumor site.

Successful resection of hepatocellular cancer not amenable to Milan criteria and durable complete remission induced by systemic polichemotherapy after development of metastases – should we think about revising the current treatment guidelines in selected patients?

ObjectivesTo refresh clinical diagnostic and therapeutic dilemmas in patients presenting with hepatocellular cancer (HCC) and to report a rare success of systemic polichemotherapy in metastatic HCC.MethodsCase report of a patient with successfully resected HCC although initially deemed inoperable according to current guidelines, and who was successfully treated by systemic polichemotherapy after development of metastatic disease, resulting in a sustained complete remission.ResultsWe describe a 71-year-old female with HCC initially treated by atypical liver resection, although not amenable to initial surgery according to current treatment guidelines, which resulted in 6 months disease-free interval. After development of pulmonary metastases, the patient was treated by systemic polichemotherapy, due to local unavailability of novel biologic agents. After 3 months of chemotherapy biochemical remission was confirmed, and after 10 months of active treatment complete radiological remission was verified according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, now exceeding 9 months in duration.ConclusionThere is an increasing body of evidence that criteria for surgical interventions in HCC should be revised and expanded, and our case is an example of such an approach. Although novel biologic therapies are not widely available in all regions of the world due to their cost, currently there are no hard recommendations for use of chemotherapy in such areas. Since this is a large problem in clinical practice, we conclude that chemotherapy should be offered to selected patients of good performance status if novel agents are unavailable.