Abstract Background: Prior data (Barron et al. Cancer Res. 2014 74:4065-77) suggests that pre-diagnostic exposure to aspirin can have significant effects on breast tumor biology and patient outcome. It has been proposed that aspirin inhibition of COX-2 may suppress lymphangiogenesis and metastasis (Karnezis et al Cancer Cell. 2014. 21:181-95). Here, we sought to recapitulate pre-diagnostic aspirin exposure in rodent models of Her2+ breast cancer and elucidate mechanisms of action. We also determined the effect of aspirin on tumor stroma, using a co-culture system of human tumor and mesenchymal stem cells (MSC). Methods: NOD/SCID mice were orthotopically implanted with Her2+ MDA-MB-231 or HCC1954 cells. 48hr later, animals began a daily low dose [30mg/kg or 120mg/kg] of aspirin, until tumors reached 250mm3. They were then resected. 3 weeks later, HCC1954 implanted animals were treated with trastuzumab (15mg/kg) and paclitaxel (5mg/kg) for 6 weeks. Primary tissues were analysed by immunohistochemistry to assess VEGF-C, -D, COX-2, LYVE1 and CD31. RNAseq was performed on tumours to identify aspirin perturbed molecular pathways. To determine the stromal response to aspirin, patient derived MSCs were cultured either alone or with HCC1954 cells and exposed to aspirin (2.5 or 7.5mM). Secreted VEGF-C was quantified. A tubule formation assay was performed to determine the impact of aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using a human angiogenesis array platform. Results: A significant delay in tumor growth was observed in both tumor models following aspirin treatment (p<.01). Assessment of metastatic progression revealed that 120 mg/kg aspirin significantly (p<.05) increased time to metastasis and reduced primary regrowth in the MDA MB 231 model (p<.01). Immunohistochemical analysis of VEGF C, D and LYVE1 showed a significant dose dependant reduction (p<.01) in both models. RNAseq pathway analysis revealed a significant over-representation of mitochondrial electron transport chain genes. Downstream factors of AMPK showed significant (p<.01) upregulation suggesting alterations in metabolism. Aspirin (7.5mM) exposure resulted in loss of VEGF-C secretion from co-cultured tumor / MSC cell populations. Conditioned media harvested following aspirin treatment limited support tubule formation. Expression of pro-angiogenic factors in HCC1954 cells showed alterations following treatment, with the greatest decrease seen in Urokinase Plasminogen Activator (-42%) and its inhibitor Serpine1 (+55%). Conclusion: We have successfully recapitulated pre-treatment aspirin response in surgical resection models of Her2+ breast cancer, with IHC analysis confirming the impact of treatment on angiogenic and lymphangiogenic factors. RNAseq analysis implicates aspirin mediated alterations in cellular metabolism. Our data further reveals increased response to aspirin in stromal cell populations. Citation Format: Ian S. Miller, Sonja Khan, Liam P. Shiels, Sudipto Das, Bruce Moran, Finbarr P. Leacy, Paul M. Loadman, Robert S. Kerbel, Darran O' Connor, Kathleen Bennett, Róisín M. Dwyer, Annette T. Byrne. Mechanistic interrogation of pre-treatment low dose aspirin effects in HER 2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1254. doi:10.1158/1538-7445.AM2017-1254
Abstract Background: One of the key events in the progression of cancer metastasis is the migration of tumor cells through the vascular endothelium. Tumors induce abnormal endothelial permeability to promote cancer cell invasion. Recently, inhibition of tumor-induced vascular permeability has been shown to attenuate metastasis in vivo (Tichet M et al. Nat Commun. 2015;6:6993). Low molecular weight heparins (LMWHs) inhibit metastasis in vivo (Klerk et al. J Clin Oncol. 2005;23(10):2130-5) but may cause bleeding complications when used at effective doses. Consequently, LMWHs are unlikely to be used for prevention of metastatic progression unless bleeding side-effects can be diminished. To address this issue, we have recently demonstrated that a LMWH/statin combination (tinzaparin and simvastatin) attenuates abnormal vascular permeability and reduces epithelial cell transmigration at concentrations unlikely to cause bleeding side-effects in vivo. (Kevane et al. Res Pract Thromb Haemost. 2017;1(1)). Here, we aim to interrogate the anti-metastatic effect of the LMWH/statin combination (tinzaparin and simvastatin) in an orthotopic surgical resection model of triple negative breast cancer implementing a LMWH/stain dose that does not increase bleeding risk. Methods: NOD/SCID mice were surgically implanted with 5x105 MDA-MB-231-LUC2 triple negative breast cancer cells in the right inguinal mammary fat pad. Tumors were allowed to grow until they reached 250mm3. Subsequently, primary tumors were surgically resected. 5 days post-resection, mice were randomized into 4 groups (group 1: 0.5% methylcellulose (vehicle), group 2: 150IU/kg TNZ, group 3: 3mg/kg SVS and group 4: TNZ (150IU/kg) and SVS (3mg/kg)) and treated daily for 4 weeks. Post-resection metastatic progression was monitored by weekly bioluminescence imaging (BLI) [IVIS Spectrum]. Animals that displayed metastatic progression or significant regrowth of primary tumour were euthanized. At time of death, apical lymph nodes and lungs were removed and fixed in 10% formalin for quantification of metastatic burden Results: Prior to study commencement it was determined that a combination of 150IU/kg TNZ and 3mg/kg SVS did not significantly increase bleeding time in a cohort of tumour naïve animals. Following primary tumor resection, BLI assessment of metastatic progression revealed that 150IU/kg TNZ, 3mg/kg SVS and combination therapy of 150IU/kg TNZ and 3mg/kg SVS significantly (P= 0.0373, P=0.0086 and P=0.0409 respectively N=11) increased the time to metastasis compared to the vehicle treated animals. Conclusion: For the first time we demonstrate that tinzaparin and simvastatin may be combined to significantly delay time to metastasis while diminishing the associated bleeding risk of LMWHs in a clinically relevant mouse model of aggressive triple negative breast cancer. Citation Format: Ian S. Miller, Liam P. Shiels, Grace Buckley, Kate Connor, Annette T. Byrne, Fionnuala Ní Ainle. Interrogating the in vivo anti-metastatic action of tinzaparin and simvastatin in an orthotopic surgical resection mouse model of triple negative breast cancer [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 2012.
Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.
Abstract Purpose: Cell-free DNA (cfDNA) analysis in plasma is an emerging technique with numerous applications in oncology. We employed cfDNA to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients and to predict outcome to bevacizumab (BVZ) combination therapy. Experimental Design: Low coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples from 74 mCRC patients participating in the CTRIAL-IE 12-16 AC-ANGIOPREDICT Phase II clinical trial (NCT01822444), prior to receiving BVZ, and analyzed for CIN (3 subclusters with low, intermediate and high CIN, respectively) and nucleosomes. For 61/74 mCRC patients, plasma samples before and after BVZ treatment were available and used for targeted methylation sequencing. A validation cohort of mCRC plasma samples (n= 24) from the University Medical Center Mannheim (UMM) was similarly profiled. Results: Based on cfDNA CIN profiles, we subtyped mCRC patients with 92.3% accuracy of sample distinction into low and high CIN clusters (cluster 1 against cluster 2 and 3), demonstrating concordance between matched plasma and tumor samples. Improved survival outcome was observed in CIN high patients. Plasma-based CIN clustering and improved survival for CIN high patients was also confirmed in the UMM cohort. NF and methylation profiles differed between CIN clusters and healthy individuals, and could reliably separate cluster 1 from cluster 2 and 3 samples (AUC=0.72 and 0.85 respectively). A large methylation score decrease after BVZ treatment was associated with improved overall survival (p = 0.013). Conclusions: cfDNA can be analyzed to predict outcome to BVZ in mCRC patients. Detection of CNAs, NFs and methylation profiles facilitated stratification of samples into CIN clusters and informed patient response to treatment. Citation Format: Ian S. Miller, Valentina Thomas, Tom Venken, Ingrid Arijs, Ana Barat, Johannes Betge, Tianzuo Zhan, Timo Gaiser, Matthias P. Ebert, Jochen Prehn, Rut Klinger, Darran P. O’Connor, Brian Brian Moulton, Verena Murphy, Ray McDermott, Brian Bird, Gregory Leonard, Liam Grogan, Anne Horgan, Nadine Schulte, Markus Moehler, Nicole Prannikar, Judith Franz-Werner, Hans-Peter Feustel, Diether Lambrechts, Annette T. Byrne. Analysis of cell free DNA to predict outcome to Bevacizumab combination therapy in metastatic colorectal cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5584.
OBJECTIVE It has been suggested that fracture of the hamulus during palatoplasty in children with cleft palate may lead to adverse otological sequelae, however, there is little evidence to support this. STUDY DESIGN AND SETTING The otological records of 42 children with repaired cleft palate (excluding submucous cleft palate) aged 8 years old or older were examined. A questionnaire regarding the incidence, treatment, and outcome of middle ear problems was completed by the parents of 68 children with repaired cleft palate, aged 9 years old or older. RESULTS There was no significant difference between children who did and did not undergo hamular fracture with regard to tympanic membrane appearance, audiometry, history of ear problems ( P = 1.000), ear infections ( P = 0.622), ventilation tube insertion ( P = 0.532), or surgery for chronic otitis media ( P = 1.000). Parents of children not undergoing hamular fracture reported a higher incidence of below normal hearing ( P = 0.023). CONCLUSION AND SIGNIFICANCE There is no evidence that hamular fracture during palatoplasty affects long‐term otological outcome in cleft palate.
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