Hypoxia Biomarkers Research Articles

Programmed cell death ligands expression in phaeochromocytomas and paragangliomas: Relationship with the hypoxic response, immune evasion and malignant behavior

ABSTRACTThe hypoxic response underlies the pathogenesis and malignant behavior of PCC/PGL. Regulation of PD-1 receptor-ligand signaling, a therapeutically actionable driver of the anti-tumor immune response, is a hypoxic-driven trait across malignancies. We evaluated the prognostic role of PD ligands in association with biomarkers of hypoxia and angiogenesis in patients with PCC/PGL.Tissue microarrays sections including consecutive cases diagnosed between 1983–2011 were stained for PD-L1 and 2, hypoxia inducible factor 1a (Hif-1a), Carbonic Anhydrase IX (CaIX), Vascular Endothelial Growth Factor-A (VEGF-A). We explored the biologic significance of PD ligands expression using gene set enrichment analysis (GSEA) on The Cancer Genome Atlas (TCGA) for PCC/PGL (n = 184).In total, 100 patients, 10% malignant, 64% PCC, 29% familial with median tumor size of 4.7 cm (range 1–14) were included. Median follow-up was 4.7 y. We found PD-L1 expression in 18% of PCC/PGL, which was independent of adverse pathological features including capsular (CI), vascular invasion (VI), necrosis (N) and expression of biomarkers of hypoxia. PD-L2 expression (16%) strongly correlated with CI, VI, N and malignant behavior (p < 0.05) and was associated with stronger Hif-1a and CaIX immunolabeling (p < 0.01). PD-L2 was predictive of shorter survival (162 versus 309 months, HR 3.1 95%CI 1.1–9.2, p = 0.02). GSEA on TGCA samples confirmed enrichment of transcripts involved in hypoxia and anti-cancer immunity.We report for the first time PD ligands expression in PCC/PGL with a distinctive prognostic, clinico-pathologic and immuno-biologic role. These findings support a potential therapeutic role for PD-1/PD-L1 targeted checkpoint inhibitors in these tumors.KEY MESSAGEThe molecular mechanisms underlying immune evasion in malignant phaeochromocytomas and paragangliomas (PCC/PGL) are poorly understood. This study demonstrates for the first time a distinctive immune-biologic and prognostic role of programmed death ligands 1 and 2 (PD-L1, PD-L2), two actionable drivers of the anti-cancer immune response. RNA-sequencing of tumor tissues reveals enrichment of transcripts relating to hypoxia and immune-exhaustion to explain the adverse clinical course observed in PD-L2 overexpressing tumors. These findings provide a rationale for the development of anti PD-1 therapies in malignant PCC/PGL.

Stratification of Pancreatic Ductal Adenocarcinoma: Combinatorial Genetic, Stromal, and Immunologic Markers.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with an immunosuppressive milieu that supports immune system evasion and disease progression. Here, we interrogated genetic, stromal, and immunologic features of PDAC to delineate impact on prognosis and means to more effectively employ immunotherapy.Experimental Design: A cohort of 109 PDAC cases annotated for overall survival was utilized as a primary discovery cohort. Gene expression analysis defined immunologic subtypes of PDAC that were confirmed in the Cancer Genome Atlas dataset. Stromal and metabolic characteristics of PDAC cases were evaluated by histologic analysis and immunostaining. Enumeration of lymphocytes, as well as staining for CD8, FOXP3, CD68, CD163, PDL1, and CTLA4 characterized immune infiltrate. Neoantigens were determined by analysis of whole-exome sequencing data. Random-forest clustering was employed to define multimarker subtypes, with univariate and multivariate analyses interrogating prognostic significance.Results: PDAC cases exhibited distinct stromal phenotypes that were associated with prognosis, glycolytic and hypoxic biomarkers, and immune infiltrate composition. Immune infiltrate was diverse among PDAC cases and enrichment for M2 macrophages and select immune checkpoints regulators were specifically associated with survival. Composite analysis with neoantigen burden, immunologic, and stromal features defined novel subtypes of PDAC that could have bearing on sensitivity to immunologic therapy approaches. In addition, a subtype with low levels of neoantigens and minimal lymphocyte infiltrate was associated with improved overall survival.Conclusions: The mutational burden of PDAC is associated with distinct immunosuppressive mechanisms that are conditioned by the tumor stromal environment. The defined subtypes have significance for utilizing immunotherapy in the treatment of PDAC. Clin Cancer Res; 23(15); 4429-40. ©2017 AACR.

Hypoxia and hypoxia response-associated molecular markers in esophageal cancer: A systematic review.

In this systematic review, the existing evidence of available hypoxia-associated molecular response biomarkers in esophageal cancer (EC) patients is summarized and set into the context of the role of hypoxia in the prediction of esophageal cancer, treatment response and treatment outcome. A systematic literature search was performed in Web of Science, MEDLINE, and PubMed databases using the keywords: hypoxia, esophagus, cancer, treatment outcome and treatment response. Eligible publications were independently evaluated by two reviewers. In total, 22 out of 419 records were included for systematic review. The described search strategy was applied weekly, with the last update being performed on April 3rd, 2017. In esophageal cancer, several (non-)invasive biomarkers for hypoxia could be identified. Independent prognostic factors for treatment response include HIF-1α, CA IX, GLUT-1 overexpression and elevated uptake of the PET-tracer 18F-fluoroerythronitroimidazole (18F-FETNIM). Hypoxia-associated molecular responses represents a clinically relevant phenomenon in esophageal cancer and detection of elevated levels of hypoxia-associated biomarkers and tends to be associated with poor treatment outcome (i.e., overall survival, disease-free survival, complete response and local control). Evaluation of tumor micro-environmental conditions, such as intratumoral hypoxia, is important to predict treatment outcome and efficacy. Promising non-invasive imaging-techniques have been suggested to assess tumor hypoxia and hypoxia-associated molecular responses. However, extensive validation in EC is lacking. Hypoxia-associated markers that are independent prognostic factors could potentially provide targets for novel treatment strategies to improve treatment outcome. For personalized hypoxia-guided treatment, safe and reliable makers for tumor hypoxia are needed to select suitable patients.

The effect of imatinib therapy on tumour cycling hypoxia, tissue oxygenation and vascular reactivity

Background: Several biomedical imaging techniques have recently been developed to probe hypoxia in tumours, including oxygen-enhanced (OE) and blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI). These techniques have strong potential for measuring both chronic and transient (cycling) changes in hypoxia, and to assess response to vascular-targeting therapies in the clinic. Methods: In this study, we investigated the use of BOLD and OE-MRI to assess changes in cycling hypoxia, tissue oxygenation and vascular reactivity to hyperoxic gas challenges, in mouse models of colorectal therapy, following treatment with the PDGF-receptor inhibitor, imatinib mesylate (Glivec). Results: Whilst no changes were observed in imaging biomarkers of cycling hypoxia (from BOLD) or chronic hypoxia (from OE-MRI), the BOLD response to carbogen-breathing became significantly more positive in some tumour regions and more negative in other regions, thereby increasing overall heterogeneity. Conclusions: Imatinib did not affect the magnitude of cycling hypoxia or OE-MRI signal, but increased the heterogeneity of the spatial distribution of BOLD MRI changes in response to gas challenges.

Programmed cell death ligands expression in pheochromocytomas (PCC) and paragangliomas (PGL): Relationship with the hypoxic response and malignant behaviour.

11597 Background: The hypoxic response underlies the pathogenesis and malignant behaviour of PCC/PGL. Regulation of PD-1 receptor-ligand signalling, a therapeutically actionable driver of the anti-tumour immune response, is a hypoxic-driven trait across malignancies. We evaluated the prognostic role of PD ligands in association with biomarkers of hypoxia and angiogenesis in patients with PCC/PGL. Methods: Tissue microarrays sections including consecutive cases of PCC/PGL diagnosed between 1983-2011 were stained for PD-L1 &amp; 2, Ki-67, hypoxia inducible factor 1a (Hif-1a), Carbonic Anhydrase IX (CaIX), Vascular Endothelial Growth Factor-A (VEGF-A). Candidate biomarkers were assessed for correlation with clinical variables including overall survival. Results: In total, 100 patients, 10% malignant, 64% PCC, 29% familial with a median tumour size of 4.7 cm (range 1-14) were included. Median follow-up was 4.7 years. PD-L1 expression was observed in 18% of cases and was independent of adverse pathological features including capsular (CI), vascular invasion (VI), necrosis (N) and expression of biomarkers of hypoxia. We observed a trend towards association with malignancy (p = 0.08). PD-L2 expression was found in 16% of tumors. PD-L2 overexpression strongly correlated with CI, VI, N (p &lt; 0.01) and malignant behaviour (p = 0.009) and was associated with stronger Hif-1a and CaIX immunolabeling (p &lt; 0.01). PD-L2 but not PD-L1 expression was predictive of shorter survival (162 versus 309 months, HR 3.1 95%CI 1.1-9.2, Log-rank p = 0.03). Gene set enrichment analysis on the TGCA PCC/PGL RNA-seq dataset (n = 184) revealed a positive correlation between PD-L2 and a number of transcripts involved in angiogenesis and immunity including Interleukin-6 (Pearson R = 0.57) and CD-8a (R = 0.56). Conclusions: We report for the first time PD-1 ligands expression in PCC/PGL with a distinctive prognostic and clinicopathologic role. These findings support a potential therapeutic role for PD-1/PD-L1 targeted checkpoint inhibitors in these tumors.

Evaluation of the R2* value in invasive ductal carcinoma with respect to hypoxic-related prognostic factors using iterative decomposition of water and fat with echo asymmetry and least-squares emission (IDEAL)

To correlate the R2* value obtained by iterative decomposition of water and fat with echo asymmetry and least-squares emission (IDEAL) with fibrotic focus (FF), microvessel density and hypoxic biomarker (HIF-1α) in breast carcinoma. Forty-two patients who were diagnosed with invasive ductal carcinoma (IDC) of the breast underwent breast MRI including IDEAL before surgery. The entire region of interest (ROI) was delineated on the R2* map, and average tumour R2* value was calculated for each ROI. Histological specimens were evaluated for the presence of FF, the microvessel density (the average microvessel density and the ratio of peripheral to central microvessel density), and the grading of HIF-1α. FF was identified in 47.6% (20/42) of IDCs. Average R2* value for IDC with FF (42.4±13.2 Hz) was significantly higher than that without FF (28.5±13.9 Hz) (P = 0.01). Spearman rank correlation suggested that the average R2* value correlated with the grade of HIF-1α and the ratio of peripheral to central microvessel density for IDCs (P < 0.001). Quantification of tumour R2* using IDEAL is associated with the presence of FF and the overexpression of HIF-1α, and may therefore be useful in predicting hypoxia of breast carcinoma. • R2* value obtained by IDEAL correlates with the overexpression of HIF-1α. • R2* value obtained by IDEAL is associated with fibrotic focus. • R2* quantification may be useful in predicting hypoxia of breast carcinoma.

18F-Fluoromisonidazole Kinetic Modeling for Characterization of Tumor Perfusion and Hypoxia in Response to Antiangiogenic Therapy.

Multiparametric imaging of tumor perfusion and hypoxia with dynamic 18F-fluoromisonidazole (18F-FMISO) PET may allow for an improved response assessment to antiangiogenic therapies. Cediranib (AZD2171) is a potent inhibitor of tyrosine kinase activity associated with vascular endothelial growth factor receptors 1, 2, and 3, currently in phase II/III clinical trials. Serial dynamic 18F-FMISO PET was performed to investigate changes in tumor biomarkers of perfusion and hypoxia after cediranib treatment. Methods: Twenty-one rats bearing HT29 colorectal xenograft tumors were randomized into a vehicle-treated control group (0.5% methylcellulose daily for 2 d [5 rats] or 7 d [4 rats]) and a cediranib-treated test group (3 mg/kg daily for 2 or 7 d; 6 rats in both groups). All rats were imaged before and after treatment, using a 90-min dynamic PET acquisition after administration of 42.1 ± 3.9 MBq of 18F-FMISO by tail vein injection. Tumor volumes were delineated manually, and the input function was image-derived (abdominal aorta). Kinetic modeling was performed using an irreversible 1-plasma 2-tissue compartmental model to estimate the kinetic rate constants K1, K1/k2, and k3-surrogates for perfusion, 18F-FMISO distribution volume, and hypoxia-mediated entrapment, respectively. Tumor-to-blood ratios (TBRs) were calculated on the last dynamic frame (80-90 min). Tumors were assessed ex vivo by digital autoradiography and immunofluorescence for microscopic visualization of perfusion (pimonidazole) and hypoxia (Hoechst 33342). Results: Cediranib treatment resulted in significant reduction of mean voxelwise 18F-FMISO TBR, K1, and K1/k2 in both the 2-d and the 7-d groups (P < 0.05). The k3 parameter was increased in both groups but reached significance only in the 2-d group. In the vehicle-treated groups, no significant change in TBR, K1, K1/k2, or k3 was observed (P > 0.2). Ex vivo tumor analysis confirmed the presence of hypoxic tumor regions that nevertheless exhibited relatively lower 18F-FMISO uptake. Conclusion:18F-FMISO kinetic modeling reveals a more detailed response to antiangiogenic treatment than a single static image is able to reveal. The reduced mean K1 reflects a reduction in tumor vascular perfusion, whereas the increased k3 reflects a rise in hypoxia-mediated entrapment of the radiotracer. However, if only late static images are analyzed, the observed reduction in 18F-FMISO uptake after treatment with cediranib may be mistakenly interpreted as a global decrease, rather than an increase, in tumor hypoxia. These findings support the use of 18F-FMISO kinetic modeling to more accurately characterize the response to treatments that have a direct effect on tumor vascularization and perfusion.

Comparison of Umbilical Serum Copeptin Relative to Erythropoietin and S100B as Asphyxia Biomarkers at Birth

Background: Birth asphyxia, estimated to account for a million neonatal deaths annually, can cause a wide variety of neurodevelopmental impairments. There is a need to develop new, swift methods to identify those neonates who would benefit from neuroprotective treatments such as hypothermia. Objectives: To examine the utility of cord serum copeptin, a stable byproduct of arginine vasopressin release, as a biomarker of birth asphyxia based on a comparison with 2 biomarkers of hypoxia and brain trauma: erythropoietin and S100B. Methods: The study population consisted of 140 singleton, term neonates: 113 controls and 27 with birth asphyxia (2/3 criteria met: umbilical artery pH <7.10, base excess ≤12 mmol/L, and 5-min Apgar score <7). All deliveries were planned vaginal, but 51 neonates were born by emergency cesarean section. Copeptin, S100B, and erythropoietin levels in umbilical artery samples were measured by immunoassays. Results: Copeptin correlated in the entire study population more strongly with umbilical artery base excess than S100B and erythropoietin, and only copeptin correlated with arterial pH. Furthermore, only copeptin levels were significantly higher in cases of birth asphyxia, and in vaginally born neonates they were found to increase as a function of labor duration. Copeptin was elevated in neonates born via vacuum extraction, whereas erythropoietin levels showed a slight increase after emergency cesarean section. Conclusions: In this study population, S100B and erythropoietin were not valid biomarkers of birth asphyxia. In contrast, our work suggests that copeptin has high potential to become a routinely used biomarker for acute birth asphyxia and neonatal distress.

Association between hypoxic volume and underlying hypoxia-induced gene expression in oropharyngeal squamous cell carcinoma

Background:Hypoxia imaging is a promising tool for targeted therapy but the links between imaging features and underlying molecular characteristics of the tumour have not been investigated. The aim of this study was to compare hypoxia biomarkers and gene expression in oropharyngeal squamous cell carcinoma (OPSCC) diagnostic biopsies with hypoxia imaged with 64Cu-ATSM PET/CT.Methods:64Cu-ATSM imaging, molecular and clinical data were obtained for 15 patients. Primary tumour SUVmax, tumour to muscle ratio (TMR) and hypoxic volume were tested for association with reported hypoxia gene signatures in diagnostic biopsies. A putative gene signature for hypoxia in OPSCCs (hypoxic volume-associated gene signature (HVS)) was derived.Results:Hypoxic volume was significantly associated with a reported hypoxia gene signature (rho=0.57, P=0.045), but SUVmax and TMR were not. Immunohistochemical staining with the hypoxia marker carbonic anhydrase 9 (CA9) was associated with a gene expression hypoxia response (rho=0.63, P=0.01). Sixteen genes were positively and five genes negatively associated with hypoxic volume (adjusted P<0.1; eight genes had adjusted P<0.05; HVS). This signature was associated with inferior 3-year progression-free survival (HR=1.5 (1.0–2.2), P=0.047) in an independent patient cohort.Conclusions:64Cu-ATSM-defined hypoxic volume was associated with underlying hypoxia gene expression response. A 21-gene signature derived from hypoxic volume from patients with OPSCCs in our study may be linked to progression-free survival.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance.

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

Multiparametric Imaging of Tumor Hypoxia and Perfusion with 18F-Fluoromisonidazole Dynamic PET in Head and Neck Cancer.

Tumor hypoxia and perfusion are independent prognostic indicators of patient outcome. We developed the methodology for and investigated the utility of multiparametric imaging of tumor hypoxia and perfusion with 18F-fluoromisonidazole (18F-FMISO) dynamic PET (dPET) in head and neck cancer. Methods: One hundred twenty head and neck cancer patients underwent 0- to 30-min 18F-FMISO dPET in a customized immobilization mask, followed by 10-min static acquisitions starting at 93 ± 6 and 160 ± 13 min after injection. A total of 248 lesions (≥2 cm3) were analyzed. Voxelwise pharmacokinetic modeling was conducted using an irreversible 1-plasma 2-tissue-compartment model to calculate surrogate biomarkers of tumor hypoxia (k3), perfusion (K1), and 18F-FMISO distribution volume. The analysis was repeated with truncated dPET datasets. Results: Substantial inter- and intratumor heterogeneity was observed for all investigated metrics. Equilibration between the blood and unbound 18F-FMISO was rapid in all tumors. 18F-FMISO distribution volume deviated from the expected value of unity, causing discrepancy between k3 maps and total 18F-FMISO uptake and reducing the dynamic range of total 18F-FMISO uptake for quantifying the degree of hypoxia. Both positive and negative trends between hypoxia and perfusion were observed in individual lesions. All investigated metrics were reproducible when calculated from a truncated 20-min dataset. Conclusion:18F-FMISO dPET provides the data necessary to generate parametric maps of tumor hypoxia, perfusion, and radiotracer distribution volume. These data clarify the ambiguity in interpreting 18F-FMISO uptake and improve the characterization of lesions. We show total acquisition times can be reduced to 20 min, facilitating the translation of 18F-FMISO dPET into the clinic.

Plasma metabolite score correlates with Hypoxia time in a newly born piglet model for asphyxia

Hypoxic-ischemic encephalopathy (HIE) secondary to perinatal asphyxia is a leading cause of mortality and acquired long-term neurologic co-morbidities in the neonate. The most successful intervention for the treatment of moderate to severe HIE is moderate whole body hypothermia initiated within 6h from birth. The objective and prompt identification of infants who are at risk of developing moderate to severe HIE in the critical first hours still remains a challenge. This work proposes a metabolite score calculated based on the relative intensities of three metabolites (choline, 6,8-dihydroxypurine and hypoxanthine) that showed maximum correlation with hypoxia time in a consolidated piglet model for neonatal hypoxia-ischemia. The metabolite score's performance as a biomarker for perinatal hypoxia and its usefulness for clinical grading and decision making have been assessed and compared to the performance of lactate which is currently considered the gold standard. For plasma samples withdrawn before and directly after a hypoxic insult, the metabolite score performed similar to lactate. However, it provided an enhanced predictive capacity at 2h after resuscitation. The present study evidences the usefulness of the metabolite score for improving the early assessment of the severity of the hypoxic insult based on serial determinations in a minimally invasive biofluid. The applicability of the metabolite score for clinical diagnosis and patient stratification for hypothermia treatment has to be confirmed in multicenter trials involving newborns suffering from HIE.

Glut-1 expression in small cervical biopsies is prognostic in cervical cancers treated with chemoradiation

Background/purposeChemoradiation (CRT) is standard therapy for locally advanced cervical cancer (LACC). However, there is a lack of biomarkers to identify patients at high relapse-risk. We examine metabolic (glucose transporter-1 [Glut-1]), hypoxic (hypoxia inducible factor [HIF-1α]; carbonic anhydrase [CA-9]) and proliferative (Ki-67) markers for prognostic utility in LACC. Materials/methods60 LACC patients treated with CRT had pre-treatment biopsies. Immunohistochemistry was performed for Glut-1, HIF-1a and CA-9, to generate a histoscore from intensity and percentage staining; and Ki-67 scored by percentage of positive cells. For each biomarker, treatment response and survival was compared between low and high-staining groups by logrank testing and multivariate analyses. ResultsHigh Glut-1 expression was associated with inferior progression-free survival (PFS), (hazard ratio [HR] 2.8, p=0.049) and overall survival (OS), (HR 5.0, p=0.011) on multifactor analysis adjusting for stage, node positivity, tumour volume and uterine corpus invasion. High Glut-1 correlated with increased risk of distant failure (HR 14.6, p=0.001) but not local failure. Low Glut-1 was associated with higher complete metabolic response rate on post-therapy positron emission tomography scan (odds ratio 3.4, p=0.048). Ki-67 was significantly associated with PFS only (HR 1.19 per 10 units increase, p=0.033). Biomarkers for hypoxia were not associated with outcome. ConclusionsHigh Glut-1 in LACC is associated with poor outcome post CRT. If prospectively validated, Glut-1 may help select patients for more intensive treatment regimens.

G6PC3, ALDOA and CS induction accompanies mir-122 down-regulation in the mechanical asphyxia and can serve as hypoxia biomarkers.

Hypoxia influences different cellular biological processes. To reveal the dynamics of hypoxia's effects on miRNA regulation in vivo, we examined the expression levels of all miRNAs in human brain and heart specimens from cases of mechanical asphyxia compared with those from cases of craniocerebral injury and hemorrhagic shock. We further validated differently expressed miRNAs in another 84 human specimens and rat models. We found that mir-122 was significantly down-regulated and that its putative targets G6PC3, ALDOA and CS were increased in the brain and cardiac tissues in cases of mechanical asphyxia compared with craniocerebral injury and hemorrhagic shock. Our data indicate that mir-122 and its targets G6PC3, ALDOA and CS play roles in the hypoxia responses that regulate glucose and energy metabolism and can serve as hypoxia biomarkers.

P07.19 Fractal structure on gadolineum-enhanced MRI scans correlates with oxidative metabolism and VEGF expression in glioblastoma

AbstractIntroduction:In glioblastoma, VEGF expression promotes angiogenesis and has been reported to correlate with contrast enhancement on MRI scans. However, clinical results of anti-angiogenic therapy seem limited and there is a need to identify glioblastoma patients that might benefit from this treatment. Fractal structure is found throughout many processes in nature, and often arises from sets of simple growth rules. In this study, we examined the contrast enhancement patterns in MRI scans of glioblastoma patients for evidence of fractal structure and correlated these to biological tumor characteristics.Materials and Methods:For 39 glioblastoma patients, volumetric T1 post-contrast MRI scans were obtained before surgical resection. For each tumor, we calculated the fractal dimension (Minkowski-Bouligand dimension) using a box-counting (cubic scaling) approach. VEGF expression was determined with use of immunohistochemistry on tissue microarrays. RNA expression microarrays from fresh frozen tissue were explored with gene set enrichment analysis (GSEA).Results:We found robust evidence for fractal structure in the contrast enhancement pattern, with an average fractal dimension of 2.17 ± 0.10, with a visually apparent split at 2.10. High fractal dimension significantly correlated with increased VEGF expression. Additionally, GSEA analysis showed a clear association between high fractal dimension and a decrease of gene sets involved in oxidative phosphorylation.Conclusion:The presence of fractal dimensionality presents a new way to analyse classic gadolineum-enhanced T1 MRI scans of glioblastoma patients. The fractal dimension of the contrast enhancement pattern provides significant information on the tumor’s metabolism and might be used as a future biomarker for hypoxia or reponse to anti-angiogenic therapy.

Tissue and imaging biomarkers for hypoxia predict poor outcome in endometrial cancer.

Hypoxia is frequent in solid tumors and linked to aggressive phenotypes and therapy resistance. We explored expression patterns of the proposed hypoxia marker HIF-1α in endometrial cancer (EC) and investigate whether preoperative functional imaging parameters are associated with tumor hypoxia. Expression of HIF-1α was explored both in the epithelial and the stromal tumor component. We found that low epithelial HIF-1α and high stromal HIF-1α expression were significantly associated with reduced disease specific survival in EC. Only stromal HIF-1α had independent prognostic value in Cox regression analysis. High stromal HIF-1α protein expression was rare in the premalignant lesions of complex atypical hyperplasia but increased significantly to invasive cancer. High stromal HIF-1α expression was correlated with overexpression of important genes downstream from HIF-1α, i.e. VEGFA and SLC2A1 (GLUT1). Detecting hypoxic tumors with preoperative functional imaging might have therapeutic benefits. We found that high stromal HIF-1α expression associated with high total lesion glycolysis (TLG) at PET/CT. High expression of a gene signature linked to hypoxia also correlated with low tumor blood flow at DCE-MRI and increased metabolism measured by FDG-PET. PI3K pathway inhibitors were identified as potential therapeutic compounds in patients with lesions overexpressing this gene signature. In conclusion, we show that high stromal HIF-1α expression predicts reduced survival in EC and is associated with increased tumor metabolism at FDG-PET/CT. Importantly; we demonstrate a correlation between tissue and imaging biomarkers reflecting hypoxia, and also possible treatment targets for selected patients.

The Utility of Serum CA9 for Prognostication in Prostate Cancer.

Carbonic anhydrase IX (CA9) catalyses the interconversion of carbon dioxide to carbonic acid and bicarbonate and is considered a putative biomarker of tumour hypoxia. We set out to evaluate the prognostic significance of CA9 in prostate cancer. Plasma samples were assessed from 68 men with high-risk localised prostate cancer treated with radical prostatectomy (RP) or radiotherapy (RT), and 20 men with castration-resistant prostate cancer (CRPC) treated with docetaxel chemotherapy between 2010 and 2012 at the Princess Margaret Cancer Centre, Canada. Of the 68 patients with high-risk localised prostate cancer, 57 underwent RP and 11 underwent RT. Baseline CA9 was not associated with recurrence or prostate-specific antigen in either group (p=0.98 and 0.20, respectively). CA9 levels before chemotherapy correlated with overall survival (r=-0.37; two-sided p=0.11). Baseline CA9 in men with CRPC may portend a more aggressive prostate cancer phenotype with poorer survival.

Abstract 1327: Gold nanorods based hypoxia-targeted photothermal cancer therapy

Abstract PURPOSE: Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. Gold nanorods (GNR), with unique dimension and optical properties, have shown superior capability for tumor photothermal ablation. This study aimed to develop a new nanoconstruct composed of gold nanorods (GNRs) conjugate to anti-carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. METHODS: GNR were synthesized according to the seed-mediated growth method with modification. Transmission electron microscopy (TEM) were used to characterize the size and shape of the particles. The GNR/anti-CAIX conjugates were prepared through a bifunctional polyethylene glycol (PEG). The conjugates were characterized by UV-Vis spectra, particle sizes and zeta potentials. The binding affinity and specificity of the GNR/anti-CAIX were evaluated with ELISA, cell adhesion assay and cell photothermal treatment. HT29 colon cancer cell line (overexpressing CAIX) and NIH3T3 cell line (no expressing CAIX) were used in our study. Near-infrared (NIR) ablation was performed on HT29 tumors in mice that treated with PBS, GNR-PEG and GNR/anti-CAIX. Biodistribution were conducted on the latter two groups. RESULTS: TEM images showed that the synthesized GNR had an aspect ratio of about 3 (8 × 24 nm). The changes of UV-vis spectra, particle sizes and zeta potentials confirmed the success of the conjugation. ELISA suggested that the number of anti-CAIX per GNR was 4.8 ± 0.3. In the cell binding assay, GNR/anti-CAIX only showed specific and high degree binding to HT29 cells but not to NIH3T3 cells. NIR laser treatment of HT29 cells resulted in significantly higher killing of GNR/anti-CAIX-targeted cells than of controls. NIR ablation of HT29 tumors in mice showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. Biodistribution study showed significant higher accumulation in tumor in the targeted-GNR group. CONCLUSIONS: GNR/anti-CAIX nanoconstructs show promise as highly efficient targeting and photothermal ablation agents for cancer treatment. GRANT SUPPORT: This work was performed under funding from NIH/NIGMS grant (1SC3GM102018-01) and NIH/NIMHD/RCMI grant (5G12RR003045-21). Citation Format: Huan Xie, Sunil Krishnan. Gold nanorods based hypoxia-targeted photothermal cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1327.

Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect of Silencing HIF-1α with Targeted Multifunctional ECO/siRNA Nanoparticles.

Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced MRI (DCE-MRI) with a biodegradable macromolecular MRI contrast agent was used to noninvasively assess the antiangiogenic effect of RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg of siRNA/kg every 3 days for 3 weeks compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silencing of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in tumor angiogenesis, glycolytic metabolism, and pH regulation. By possessing the ability to elicit a multifaceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment.

Aberrant methylation of MUC1 and MUC4 promoters are potential prognostic biomarkers for pancreatic ductal adenocarcinomas.

Pancreatic cancer is still a disease of high mortality despite availability of diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic neoplasms. MUC1 and MUC4 are high molecular weight transmembrane mucins. These are overexpressed in many carcinomas, and high expression of these molecules is a risk factor associated with poor prognosis. We evaluated the methylation status of MUC1 and MUC4 promoter regions in pancreatic tissue samples from 169 patients with various pancreatic lesions by the methylation specific electrophoresis (MSE) method. These results were compared with expression of MUC1 and MUC4, several DNA methylation/demethylation factors (e.g. ten-eleven translocation or TET, and activation-induced cytidine deaminase or AID) and CAIX (carbonic anhydrase IX, as a hypoxia biomarker). These results were also analyzed with clinicopathological features including time of overall survival of PDAC patients. We show that the DNA methylation status of the promoters of MUC1 and MUC4 in pancreatic tissue correlates with the expression of MUC1 and MUC4 mRNA. In addition, the expression of several DNA methylation/demethylation factors show a significant correlation with MUC1 and MUC4 methylation status. Furthermore, CAIX expression significantly correlates with the expression of MUC1 and MUC4. Interestingly, our results indicate that low methylation of MUC1 and/or MUC4 promoters correlates with decreased overall survival. This is the first report to show a relationship between MUC1 and/or MUC4 methylation status and prognosis. Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with PDAC.