Hypoxia Biomarkers Research Articles

Administration of Hypoxia-Activated Prodrug Evofosfamide after Conventional Adjuvant Therapy Enhances Therapeutic Outcome and Targets Cancer-Initiating Cells in Preclinical Models of Colorectal Cancer.

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs.Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia.Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide.Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116-27. ©2018 AACR.

Potential biomarkers of tissue hypoxia during acute hemodilutional anemia in cardiac surgery: A prospective study to assess tissue hypoxia as a mechanism of organ injury

Hemodilutional anemia is associated with acute kidney injury (AKI) and mortality in patients undergoing cardiac surgery by mechanisms that may include tissue hypoxia. Our hypothesis was to assess if changes in the potential hypoxic biomarkers, including methemoglobin and erythropoietin, correlated with a decrease in hemoglobin (Hb) concentration following hemodilution on cardiopulmonary bypass (CPB). Arterial blood samples were taken from patients (n = 64) undergoing heart surgery and CPB at baseline, during CPB, following CPB, and in the intensive care unit (ICU). Potential hypoxic biomarkers were measured, including methemoglobin, plasma Hb, and erythropoietin. Data were analyzed by repeated measures one-way analysis of variance on ranks and linear regression. Hemoglobin levels decreased following CPB and methemoglobin increased in the ICU (P < 0.001 for both). No correlation was observed between the change in Hb and methemoglobin (P = 0.23). By contrast, reduced Hb on CPB correlated with increased lactate, reduced pH, and increased erythropoietin levels following CPB (P ≤ 0.004 for all). Increased plasma Hb (P < 0.001) also correlated with plasma erythropoietin levels (P < 0.001). These data support the hypothesis that erythropoietin rather than methemoglobin is a potential biomarker of anemia-induced tissue hypoxia. The observed relationships between decreased Hb during CPB and the increase in lactate, reduced pH, and increase in erythropoietin levels suggest that early changes in plasma erythropoietin may be a pragmatic early biomarker of anemia-induced renal hypoxia. Further study is required to determine if anemia-induced increases in erythropoietin may predict AKI in patients undergoing cardiac surgery. www.clinicaltrials.gov (NCT01883713). Registered 21 June 2013.

Impact of Oxygen Concentration on Metabolic Profile of Human Placenta-Derived Mesenchymal Stem Cells As Determined by Chemical Isotope Labeling LC-MS.

The placenta resides in a physiologically low oxygen microenvironment of the body. Hypoxia induces a wide range of stem cell cellular activities. Here, we report a workflow for exploring the role of physiological (hypoxic, 5% oxygen) and original cell culture (normoxic, 21% oxygen) oxygen concentrations in regulating the metabolic status of human placenta-derived mesenchymal stem cells (hPMSCs). The general biological characteristics of hPMSCs were assessed via a variety of approaches such as cell counts, flow cytometry and differentiation study. A sensitive 13C/12C-dansyl labeling liquid chromatography-mass spectrometry (LC-MS) method targeting the amine/phenol submetabolome was used for metabolic profiling of the cell and corresponding culture supernatant. Multivariate and univariate statistical analyses were used to analyze the metabolomics data. hPMSCs cultured in hypoxia display smaller size, higher proliferation, greater differentiation ability and no difference in immunophenotype. Overall, 2987 and 2860 peak pairs or metabolites were detected and quantified in hPMSCs and culture supernatant, respectively. Approximately 86.0% of cellular metabolites and 84.3% of culture supernatant peak pairs were identified using a dansyl standard library or matched to metabolite structures using accurate mass search against human metabolome libraries. The orthogonal partial least-squares discriminant analysis (OPLS-DA) showed a clear separation between the hypoxic group and the normoxic group. Ten metabolites from cells and six metabolites from culture supernatant were identified as potential biomarkers of hypoxia. This study demonstrated that chemical isotope labeling LC-MS can be used to reveal the role of oxygen in the regulation of hPMSC metabolism, whereby physiological oxygen concentrations may promote arginine and proline metabolism, pantothenate and coenzyme A (CoA) biosynthesis, and alanine, aspartate and glutamate metabolism.

Based on the Metabolomic Approach the Energy Metabolism Responses of Oriental River Prawn Macrobrachium nipponense Hepatopancreas to Acute Hypoxia and Reoxygenation.

Hypoxia represents a major physiological challenge for prawns and is a problem in aquaculture. Therefore, an understanding of the metabolic response mechanism of economically important prawn species to hypoxia and re-oxygenation is essential. However, little is known about the intrinsic mechanisms by which the oriental river prawn Macrobrachium nipponense copes with hypoxia at the metabolic level. In this study, we conducted gas chromatography-mass spectrometry-based metabolomics studies and assays of energy metabolism-related parameters to investigate the metabolic mechanisms in the hepatopancreas of M. nipponense in response to 2.0 O2/L hypoxia for 6 and 24 h, and reoxygenation for 6 h following hypoxia for 24 h. Prawns under hypoxic stress displayed higher glycolysis-related enzyme activities and lower mRNA expression levels of aerobic respiratory enzymes than those in the normoxic control group, and those parameters returned to control levels in the reoxygenated group. Our results showed that hypoxia induced significant metabolomic alterations in the prawn hepatopancreas within 24 h. The main metabolic alterations were depletion of amino acids and 2-hydroxybutanoic acid and accumulation of lactate. Further, the findings indicated that hypoxia disturbed energy metabolism and induced antioxidant defense regulation in prawns. Surprisingly, recovery from hypoxia (i.e., reoxygenation) significantly affected 25 metabolites. Some amino acids (valine, leucine, isoleucine, lysine, glutamate, and methionine) were markedly decreased compared to the control group, suggesting that increased degradation of amino acids occurred to provide energy in prawns at reoxygenation conditions. This study describes the acute metabolomic alterations that occur in prawns in response to hypoxia and demonstrates the potential of the altered metabolites as biomarkers of hypoxia.

Levels of Endocan, Angiopoietin-2, and Hypoxia-Inducible Factor-1a in Patients with Autosomal Dominant Polycystic Kidney Disease and Different Levels of Renal Function

Background: Endothelial dysfunction leading to unbalanced vasoconstriction and ischemia of renal parenchyma is increasingly proposed as an alternative pathway of renal damage in autosomal dominant polycystic kidney disease (ADPKD). However, human studies investigating the evolution of such phenomena are limited. This study investigated the levels of emerging biomarkers of endothelial function, angiogenesis and hypoxia, in ADPKD patients with different renal function. Methods: The study population consisted of three groups: 26 ADPKD patients with impaired renal function (Group A; estimated glomerular filtration rate [eGFR] 45–70 mL/min/1.73 m²), 26 ADPKD patients with preserved renal function (Group B; eGFR >70 mL/min/1.73 m²), and 26 age- and sex-matched controls with no history of renal disease. Circulating levels of endocan (endothelial cell-specific molecule-1) angiopoietin-2, and hypoxia-inducible factor-1a (HIF-1a) were determined by enzyme-linked immunosorbent assay techniques. Results: Patients in Group A had significantly higher levels of endocan (7.17 ± 0.43 ng/mL), angiopoietin-2 (5,595.43 ± 3,390), and HIF-1a (163.68 ± 37.84 pg/mL) compared to patients in Group B (6.86 ± 0.59 ng/mL, p = 0.017, 3,854.41 ± 3,014.30, p = 0.018, 136.84 ± 42.10 pg/mL, p = 0.019 respectively) or controls (4.83 ± 0.69 ng/mL, 1,069 ± 427.88 pg/mL, 70.20 ± 17.49 pg/mL, p < 0.001 for all comparisons). Of note, patients in Group B had also higher levels of all markers compared to controls (p < 0.001) despite having similar renal function. In correlation analyses within ADPKD patients, we noted strong correlations of all studied markers with asymmetric dimethylarginine (ADMA; endocan r = 0.908, p < 0.001, angiopoietin-2 r = 0.983, p < 0.001 and HIF-1a r = 0.998, p < 0.001), and only weak or modest correlations with eGFR. Conclusions: This study suggests that endothelial dysfunction causing microcirculatory changes, linked to angiogenesis and hypoxia, may come early in the course of ADPKD and could be a key regulator of renal injury progression.

Hypoxia gene expression signatures as predictive biomarkers for personalising radiotherapy.

Hypoxia is a generic micro-environmental factor of solid tumours. High levels of hypoxia lead to resistance to radiotherapy, which can be targeted by adding hypoxia-modifying therapy to improve clinical outcomes. Not all patients benefit from hypoxia-modifying therapy, and there is a need for biomarkers to enable progression to biologically personalised radiotherapy. Gene expression signatures are a relatively new category of biomarkers that can reflect tumour hypoxia. This article reviews the published hypoxia gene signatures, summarising their development and validation. The challenges of gene signature derivation and development, and advantages and disadvantages in comparison with other hypoxia biomarkers are also discussed. Current evidence supports investment in gene signatures as a promising hypoxia biomarker approach for clinical utility.

Exosomal microRNA profiling to identify hypoxia-related biomarkers in prostate cancer.

Hypoxia and expression of hypoxia-related biomarkers are associated with disease progression and treatment failure in prostate cancer (PCa). We have reported that exosomes (nanovesicles of 30-150 nm in diameter) secreted by human PCa cells under hypoxia promote invasiveness and stemness in naïve PCa cells. Here, we identified the unique microRNAs (miRNAs) loaded in exosomes secreted by PCa cells under hypoxia. Using TaqMan® array microRNA cards, we analyzed the miRNA profile in exosomes secreted by human PCa LNCaP cells under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions. We identified 292 miRNAs loaded in both ExoHypoxic and ExoNormoxic. The top 11 miRNAs with significantly higher level in ExoHypoxic compared to ExoNormoxic were miR-517a, miR-204, miR-885, miR-143, miR-335, miR-127, miR-542, miR-433, miR-451, miR-92a and miR-181a; and top nine miRNA with significantly lower expression level in ExoHypoxic compared to ExoNormoxic were miR-521, miR-27a, miR-324, miR-579, miR-502, miR-222, miR-135b, miR-146a and miR-491. Importantly, the two differentially expressed miRNAs miR-885 (increased expression) and miR-521 (decreased expression) showed similar expression pattern in exosomes isolated from the serum of PCa patients compared to healthy individuals. Additionally, miR-204 and miR-222 displayed correlated expression patterns in prostate tumors (Pearson R = 0.66, p < 0.0001) by The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) genomic dataset analysis. Overall, the present study identified unique miRNAs with differential expression in exosomes secreted from hypoxic PCa cells and suggests their potential usefulness as a biomarker of hypoxia in PCa patients.

Umbilical cord blood Erythropoietin as a biomarker of subacute and chronic hypoxia in deliveries complicated by meconium stained amniotic fluid

Background: The presence of meconium stained amniotic fluid (MSAF) during labour is often associated with a lot of adverse outcomes and historically has been considered to be a predictor of bad neonatal outcome. It is associated with fetal hypoxia and leads to emergency C/S, instrumental vaginal delivery for fetal distress, low Apgar score at birth, meconium aspiration syndrome and neuro-developmental handicaps. Umbilical cord serum erythropoietin levels have been claimed to be elevated in response to fetal hypoxia. Objective: To evaluate the umbilical cord serum erythropoietin levels in neonates born through MSAF and to determine whether it is influenced by gestational age. Study Design: Prospective case control study. Materials & Methods: Women with term uncomplicated singlet on pregnancies at gestational age between (37-41 +6 ) weeks admitted to the labour room in the active phase of labour were enrolled to participate in this study. The studied population has been divided into two groups, women with thick meconium stained amniotic fluid (study group) and women with clear amniotic fluid (control group). Umbilical cord serum Erythropoietin levels will be measured for both groups by ELISA, after delivery and Apgar score will be assessed for neonates of both groups then statistical analysis will be performed. Results: A significant relationship was found between MSAF and elevated umbilical cord serum erythropoietin levels since the p value was P < 0.001. Apgar score was lower significantly after 1 minute in study group, but lower none significantly after 5 minutes, as P <0.001and 0.109 respectively. Conclusions: The elevated umbilical cord serum Erythropoietin levels in neonates born through MSAF suggest subacute or chronic fetal Hypoxia, independent of gestational age.

Characteristics of new bioreductive fluorescent probes based on the xanthene fluorophore: Detection of nitroreductase and imaging of hypoxic cells

Hypoxia is a common feature of many solid tumors and is known to cause the resistance to chemotherapy and radiotherapy. Nitroreductase (NTR), a common biomarker of hypoxia, is an attractive target for the design of therapeutics and imaging agents targeting hypoxia. Under hypoxic condition, nitroreductase can reduce an aromatic nitro group conjugated to a drug or fluorophore to amine, which leads to release a free drug or fluorophore through the subsequent 1,6-rearrangment elimination. This type of 1,6-rearrangement elimination to release a drug or fluorophore has been widely used in the development of activity-based “turn on” fluorescent probes as well as targeted therapeutics such as antibody-drug conjugates (ADCs) and small molecule-drug conjugates (SMDCs). In this paper, novel “turn on” NTR-responsive fluorescent probes diversely linked to a 4-nitrobenzyl moiety via ether, carbonate, amine and carbamate linkages have been developed. The effect of variation in linkage on the release of fluorophore has been studied by the analysis of spectroscopic properties, temperature and pH stability, kinetics and concentration-dependency of the probes during NTR reaction. Fluorescent probes with an ether (10) or a carbamate (16) linkage showed biocompatible nature, high sensitivity (working below 1 μM of concentration) and strong fluorescence emission in the presence of NTR whereas they showed very low quantum yields in the absence of NTR. Finally, the probes (10, 16) were successfully applied to in vitro imaging of hypoxic cancer cells.

Imageable Biomarkers for Radiotherapy Response.

Ideally, each patient with a malignancy who is eligible for radiation therapy should receive the most tumoricidal form of this this treatment with the lowest possible risk of toxicity. To overcome radiotherapy resistance, some patients would benefit from a more aggressive approach. This could be treatment intensification, for example by acceleration of the treatment to prevent the negative effects of accelerated tumor cell proliferation, or by boosting certain areas to specifically address intrinsic radioresistance, or a combination of radiotherapy with, for example, a hypoxic cell sensitizer or chemotherapy to reduce the radiotherapy resistance caused by hypoxia. For some patients, one of these approaches can be beneficial but for others could lead to unacceptable side effects. Therefore, it is highly desirable to make the selection upfront. The use of imageable biomarkers could be the key to a more patient-tailored treatment. Different biomarkers for hypoxia and proliferation that could be valuable for radiotherapy are discussed here, including their mechanism, the imaging procedure, quantification, and the value of the results.

Abstract A041: Gene expression alteration in chronic hypoxic PANC-1 pancreatic cancer cell line

Abstract Background: Cancer cells grow uncontrollably by disregarding the normal rule of cell division; the resulting overcrowding of cancer cells leads to a decrease in oxygen availability, which is known as hypoxia. The role of tumor hypoxia has become a main focus in cancer research since it is a driving force in promoting tumor progression, malignancy, and resistance to therapy. Hypoxia is a prominent feature of the tumor microenvironment of pancreatic tumors. Objective: The main purpose of this study was to identify the changes in gene expression associated with chronic hypoxia in PANC-1 pancreatic cancer cell line. Methodology: Pancreatic cancer cells (PANC-1) were exposed to 8-hour hypoxic episodes (&amp;lt;1% oxygen) three times a week for a total of 40 episodes. The alterations in gene expression were examined using the real-time based PCR array technology after 20 and 40 episodes of hypoxia and compared to normoxic cells. Chemo-resistance of hypoxic cells toward doxorubicin was assessed using MTT cell proliferation assay. Cell migration was evaluated using wound healing assay. Results: Noticeable gene expression changes were identified after 20 and 40 episodes of hypoxia. No changes were common in both stages, with most genes rebounding at the level of 40 episodes. Notably, genes HIF1AN, HMOX-1, and PKM have been remarkably upregulated by 6.9, 4.5, and 3.4 fold, respectively, after 20 hypoxic shots. The half-maximal inhibitory concentration (IC50) of doxorubicin in PANC-1 cells has increased by approximately 2.7 fold after 20 episodes of hypoxia and increased by approximately 3.6 fold after 40 episodes compared to normoxic cells. Conclusion: This study provided evidence that exposing cells to prolonged periods of hypoxia (weeks) results in different expression changes than those induced by short-term hypoxia (&amp;lt;72 hours). It also approved induction of chemo-resistance by chronic hypoxia. Finally, it proposed HIF1AN, HMOX-1, and PKM as a potential biomarkers for hypoxia in pancreatic cancer and as main players in the cellular response to chronic hypoxia. Citation Format: Malek A. Zihlif, Layaly Hisham Younis Hisham Younis, Ahmad Sharab Sharab. Gene expression alteration in chronic hypoxic PANC-1 pancreatic cancer cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A041.

Cell Death, Inflammation, Tumor Burden, and Proliferation Blood Biomarkers Predict Lung Cancer Radiotherapy Response and Correlate With Tumor Volume and Proliferation Imaging

IntroductionThere is an unmet need to develop noninvasive biomarkers to stratify patients in drug-radiotherapy trials. In this pilot study we investigated lung cancer radiotherapy response and toxicity blood biomarkers and correlated findings with tumor volume and proliferation imaging. Patients and MethodsBlood samples were collected before and during (day 21) radiotherapy. Twenty-six cell-death, hypoxia, angiogenesis, inflammation, proliferation, invasion, and tumor-burden biomarkers were evaluated. Clinical and laboratory data were collected. Univariate analysis was performed on small-cell and non–small-cell lung cancer (NSCLC) whereas multivariate analysis focused on NSCLC. ResultsBlood samples from 78 patients were analyzed. Sixty-one (78.2%) harbored NSCLC, 48 (61.5%) received sequential chemoradiotherapy. Of tested baseline biomarkers, undetectable interleukin (IL)-1b (hazard ratio [HR], 4.02; 95% confidence interval [CI], 2.04-7.93; P < .001) was the only significant survival covariate. Of routinely collected laboratory tests, high baseline neutrophil count was a significant survival covariate (HR, 1.07; 95% CI, 1.02-1.11; P = .017). Baseline IL-1b and neutrophil count were prognostic for survival in a multivariate model. The addition of day-21 cytokeratin-19 antigen modestly improved this model's survival prediction (concordance probability, 0.75-0.78). Chemotherapy (P < .001) and baseline keratinocyte growth factor (P = .019) predicted acute esophagitis, but only chemotherapy remained significant after Bonferroni correction. Baseline angioprotein-1 and hepatocyte growth factor showed a direct correlation with tumor volume whereas changes in vascular cell adhesion molecule 1 showed significant correlations with 18F-fluorothymidine (FLT) positron emission tomography (PET). ConclusionSelect biomarkers are prognostic after radiotherapy in this lung cancer series. The correlation between circulating biomarkers and 18F-FLT PET is shown, to our knowledge for the first time, highlighting their potential role as imaging surrogates.

The Effect of Neoadjuvant Androgen Deprivation Therapy on Tumor Hypoxia in High-Grade Prostate Cancer: A 18F-MISO PET/MRI Imaging Study

Tumor hypoxia has been associated with a higher risk of biochemical recurrence in prostate cancer (PCa) patients treated with curative radiotherapy (RT). Fluoromisonidazole (18F-MISO) is a promising PET tracer tested in some cancer types as a tool to identify and target hypoxic areas. In this prospective study we assessed the occurrence of hypoxia and evaluated the correlation between tissue and imaging hypoxia-biomarkers before and after 3 months of neoadjuvant androgen deprivation therapy (nADT) in high-grade PCa patients treated with curative RT. Eleven non-metastatic high-risk PCa patients with a bioptic Gleason score (GS) ≥ 8 underwent two 18F-MISO hybrid PET/MRI studies before and after 3 months of nADT, respectively. In all patients, a 18F-Choline (CHO) PET/CT was realized for initial staging. 18F-MISO target–to–blood pool ratio (TBR=SUVmax/SUVmean blood) and CHO SUVmax were estimated for each patient based on a sextant repartition of the prostate gland, while the CHO metabolic tumor volume (MTV) was measured for all macroscopically visible tumor lesions. Immunohistochemistry for tissue hypoxia- and proliferation-related biomarkers (EGFR, CAIX, VEGF-A, HIF-1alpha, GLUT1 and Ki67) was performed in all the biopsies cores with the highest GS. Correlations between tissue biomarkers and 18F-MISO TBR, CHO SUVmax and MTV values, mpMRI Prostate Imaging Reporting and Data System (PIRADS) scores, and GS repartition in the whole prostate gland before nADT initiation were made using non parametric tests. The pre- and post-treatment MISO TBR was compared in all biopsy-positives areas by repeated measures ANOVA, controlling for the repeated measures in different zones of an individual patients as “between subjects” factor. A significant focal 18F-MISO uptake (SUV>1.4) was visible in 7 patients (21 zones) corresponding in 5 cases to the highest GS lesion. Among the 21 zones with a significant initial uptake, only 6 zones (in 3 cases the highest GS lesion) maintained an uptake higher than 1.4 after nADT. Mean 18F-MISO TBR before nADT (1.26±0.4) was significantly higher than after nADT (1.06±0.4) (p=0.005). A statistically significant relation was found between GS and 18F-MISO TBR (.351, p=0.018), between GS and CHO SUVmax (.570, p<0.001), between GS and PIRADS (.494, p=0.001), and between CHO SUVmax and PIRADS (.346, p=0.02). No statistically significant correlation was found between MISO imaging and tissue hypoxia biomarkers. 18F-MISO PET imaging showed variable uptake in PCa, associated with GS and lowering significantly after 3 months of nADT. These findings suggest the existence of a hypoxic microenvironment in PCa and further support the re-oxygenation role of the nADT phase and its potential role in overcoming tumor resistance in high-risk PCa.

18F]Fluoromisonidazole PET in rectal cancer

BackgroundThere is an increasing interest in developing predictive biomarkers of tissue hypoxia using functional imaging for personalised radiotherapy in patients with rectal cancer that are considered for neoadjuvant chemoradiotherapy (CRT). The study explores [18F]fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) scans for predicting clinical response in rectal cancer patients receiving neoadjuvant CRT.MethodsPatients with biopsy-proven rectal adenocarcinoma were imaged at 0–45 min, 2 and 4 h, at baseline and after 8–10 fractions of CRT (week 2). The first 6 patients did not receive an enema (the non-enema group) and the last 4 patients received an enema before PET-CT scan (the enema group). [18F]FMISO production failed on 2 occasions. Static PET images at 4 h were analysed using tumour-to-muscle (T:M) SUVmax and tumour-to-blood (T:B) SUVmax. The 0–45 min dynamic PET scans were analysed using Casciari model to report hypoxia and perfusion. Akaike information criteria (AIC) were used to compare data fittings for different pharmacokinetic models. Pathological tumour regression grade was scored using American Joint Committee on Cancer (AJCC) 7.0. Shapiro-Wilk test was used to evaluate the normality of the data.ResultsFive out of eleven (5/11) patients were classed as good responders (AJCC 0/1 or good clinical response) and 6/11 as poor responders (AJCC 2/3 or poor clinical response). The median T:M SUVmax was 2.14 (IQR 0.58) at baseline and 1.30 (IQR 0.19) at week 2, and the corresponding median tumour hypoxia volume was 1.08 (IQR 1.31) cm3 and 0 (IQR 0.15) cm3, respectively. The median T:B SUVmax was 2.46 (IQR 1.50) at baseline and 1.61 (IQR 0.14) at week 2, and the corresponding median tumour hypoxia volume was 5.68 (IQR 5.86) cm3 and 0.76 (IQR 0.78) cm3, respectively. For 0–45 min tumour modelling, the median hypoxia was 0.92 (IQR 0.41) min−1 at baseline and 0.70 (IQR 0.10) min−1 at week 2. The median perfusion was 4.10 (IQR 1.71) ml g−1 min−1 at baseline and 2.48 (IQR 3.62) ml g−1 min−1 at week 2. In 9/11 patients with both PET scans, tumour perfusion decreased in non-responders and increased in responders except in one patient. None of the changes in other PET parameters showed any clear trend with clinical outcome.ConclusionsThis pilot study with small number of datasets revealed significant challenges in delivery and interpretation of [18F]FMISO PET scans of rectal cancer. There are two principal problems namely spill-in from non-tumour tracer activity from rectal and bladder contents. Emphasis should be made on reducing spill-in effects from the bladder to improve data quality. This preliminary study has shown fundamental difficulties in the interpretation of [18F]FMISO PET scans for rectal cancer, limiting its clinical applicability.

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Value of combining dynamic contrast enhanced ultrasound and optoacoustic tomography for hypoxia imaging

Optoacoustic imaging (OAI) can detect haemoglobin and assess its oxygenation. However, the lack of a haemoglobin signal need not indicate a lack of perfusion. This study uses a novel method to assist the co-registration of optoacoustic images with dynamic contrast enhanced ultrasound (DCE-US) images to demonstrate, in preclinical tumour models, the value of combining haemoglobin imaging with a perfusion imaging method, showing that a lack of a haemoglobin signal does not necessarily indicate an absence of perfusion. DCE-US was chosen for this particular experiment because US is extremely sensitive to microbubble contrast agents and because microbubbles, like red blood cells but unlike currently available optical contrast agents, do not extravasate. Significant spatial correlations were revealed between the DCE-US properties and tumour blood-oxygen saturation and haemoglobin, as estimated using OAI. It is speculated that DCE-US properties could be applied as surrogate biomarkers for hypoxia when planning clinical radiotherapy or chemotherapy.

Targeting Hypoxia to Improve Non-Small Cell Lung Cancer Outcome.

Oxygen deprivation (hypoxia) in non-small cell lung cancer (NSCLC) is an important factor in treatment resistance and poor survival. Hypoxia is an attractive therapeutic target, particularly in the context of radiotherapy, which is delivered to more than half of NSCLC patients. However, NSCLC hypoxia-targeted therapy trials have not yet translated into patient benefit. Recently, early termination of promising evofosfamide and tarloxotinib bromide studies due to futility highlighted the need for a paradigm shift in our approach to avoid disappointments in future trials. Radiotherapy dose painting strategies based on hypoxia imaging require careful refinement prior to clinical investigation. This review will summarize the role of hypoxia, highlight the potential of hypoxia as a therapeutic target, and outline past and ongoing hypoxia-targeted therapy trials in NSCLC. Evidence supporting radiotherapy dose painting based on hypoxia imaging will be critically appraised. Carefully selected hypoxia biomarkers suitable for integration within future NSCLC hypoxia-targeted therapy trials will be examined. Research gaps will be identified to guide future investigation. Although this review will focus on NSCLC hypoxia, more general discussions (eg, obstacles of hypoxia biomarker research and developing a framework for future hypoxia trials) are applicable to other tumor sites.

A Probe for the Detection of Hypoxic Cancer Cells.

Hypoxia is a common feature of tumor cells. Nitroreductase (NTR), a common biomarker of hypoxia, has been widely used to evaluate the extent of tumor hypoxia. In this study, three fluorescent probes (FBN-1-3) were synthesized to monitor the extent of hypoxia in cancer cells in real time. FBN-1-3 were composed of a fluorescein analogue and one of three different aromatic nitro groups. Of these probes, FBN-1 showed excellent sensitivity and selectivity in detecting hypoxia via a reduction in O2 concentration. Confocal fluorescence imaging and flow cytometry demonstrated that HepG-2, A549, and SKOV-3 cells incubated with FBN-1 under reduced oxygen conditions showed significantly enhanced fluorescence. A mouse HepG-2 tumor model confirmed that FBN-1 responds rapidly to NTR and can be used to evaluate the degree of tumor hypoxia. The changes in intra- and extracellular NTR in tumor cells were also concurrently monitored, which did not reveal a link between NTR concentration and degree of hypoxia. Our work provides a functional probe for tumor hypoxia, and our results suggest the fluorescent response of our probe is due to a decrease in O2 concentration, and not NTR concentration.

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.