A phase 1 'window-of-opportunity' trial testing evofosfamide (TH-302), a tumour-selective hypoxia-activated cytotoxic prodrug, with preoperative chemoradiotherapy in oesophageal adenocarcinoma patients.

BACKGROUND: Neo-adjuvant chemoradiotherapy followed by surgery is the standard treatment with curative intent for oesophageal cancer patients, with 5-year overall survival rates up to 50 %. However, patients' quality of life is severely compromised by oesophagectomy, and eventually many patients die due to metastatic disease. Most solid tumours, including oesophageal cancer, contain hypoxic regions that are more resistant to chemoradiotherapy. The hypoxia-activated prodrug evofosfamide works as a DNA-alkylating agent under these hypoxic conditions, which directly kills hypoxic cancer cells and potentially minimizes resistance to conventional therapy. This drug has shown promising results in several clinical studies when combined with chemotherapy. Therefore, in this phase I study we investigate the safety of evofosfamide added to the chemoradiotherapy treatment of oesophageal cancer. METHODS/DESIGN: A phase I, non-randomized, single-centre, open-label, 3 + 3 trial with repeated hypoxia PET imaging, will test the safety of evofosfamide in combination with neo-adjuvant chemoradiotherapy in potentially resectable oesophageal adenocarcinoma patients. Investigated dose levels range from 120 mg/m2 to 340 mg/m2. Evofosfamide will be administered one week before the start of chemoradiotherapy (CROSS-regimen) and repeated weekly up to a total of six doses. PET/CT acquisitions with hypoxia tracer (18)F-HX4 will be made before and after the first administration of evofosfamide, allowing early assessment of changes in hypoxia, accompanied with blood sampling to measure hypoxia blood biomarkers. Oesophagectomy will be performed according to standard clinical practice. Higher grade and uncommon non-haematological, haematological, and post-operative toxicities are the primary endpoints according to the CTCAEv4.0 and Clavien-Dindo classifications. Secondary endpoints are reduction in hypoxic fraction based on (18)F-HX4 imaging, pathological complete response, histopathological negative circumferential resection margin (R0) rate, local and distant recurrence rate, and progression free and overall survival. DISCUSSION: This is the first clinical trial testing evofosfamide in combination with chemoradiotherapy. The primary objective is to determine the dose limiting toxicity of this combined treatment and herewith to define the maximum tolerated dose and recommended phase 2 dose for future clinical studies. The addition of non-invasive repeated hypoxia imaging ('window-of-opportunity') enables us to identify the biologically effective dose. We believe this approach could also be used for other hypoxia targeted drugs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02598687 .

Pre-transplant kidney quality evaluation using photoacoustic imaging during normothermic machine perfusion.

Due to the shortage of kidneys donated for transplantation, surgeons are forced to use the organs with an elevated risk of poor function or even failure. Although the existing methods for pre-transplant quality evaluation have been validated over decades in population cohort studies across the world, new methods are needed as long as delayed graft function or failure in a kidney transplant occurs. In this study, we explored the potential of utilizing photoacoustic (PA) imaging during normothermic machine perfusion (NMP) as a means of evaluating kidney quality. We closely monitored twenty-two porcine kidneys using 3D PA imaging during a two-hour NMP session. Based on biochemical analyses of perfusate and produced urine, the kidneys were categorized into 'non-functional' and 'functional' groups. Our primary focus was to quantify oxygenation (sO2) within the kidney cortical layer of depths 2 mm, 4 mm, and 6 mm using two-wavelength PA imaging. Next, receiver operating characteristic (ROC) analysis was performed to determine an optimal cortical layer depth and time point for the quantification of sO2 to discriminate between functional and non-functional organs. Finally, for each depth, we assessed the correlation between sO2 and creatinine clearance (CrCl), oxygen consumption (VO2), and renal blood flow (RBF). We found that hypoxia of the renal cortex is associated with poor renal function. In addition, the determination of sO2 within the 2 mm depth of the renal cortex after 30 min of NMP effectively distinguishes between functional and non-functional kidneys. The non-functional kidneys can be detected with the sensitivity and specificity of 80% and 85% respectively, using the cut-off point of sO2 < 39%. Oxygenation significantly correlates with RBF and VO2 in all kidneys. In functional kidneys, sO2 correlated with CrCl, which is not the case for non-functional kidneys. We conclude that the presented technique has a high potential for supporting organ selection for kidney transplantation.

Improved Multimodal Tumor Necrosis Imaging with IRDye800CW-DOTA Conjugated to an Albumin-Binding Domain.

PURPOSE: To assess our improved NACA for the detection of tumor necrosis. METHODS: We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [18F]FDG-PET and bioluminescence images for complementary detection of tumor viability. RESULTS: We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [111In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [111In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [18F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [111In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors. CONCLUSIONS: Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.

In Vivo Evaluation of Gallium-68-Labeled IRDye800CW as a Necrosis Avid Contrast Agent in Solid Tumors.

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Necrosis binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate: a consequence from cyanine-labeling of small molecules.

BACKGROUND: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. RESULTS: Binding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. CONCLUSION: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.

In Vivo Evaluation of Indium-111-Labeled 800CW as a Necrosis-Avid Contrast Agent.

PURPOSE: Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. PROCEDURES: We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [111In]In-DOTA-PEG4-800CW. We then investigated specific [111In]In-DOTA-PEG4-800CW uptake by dead cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated [111In]In-DOTA-PEG4-800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. RESULTS: We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [111In]In-DOTA-PEG4-800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. CONCLUSIONS: [111In]In-DOTA-PEG4-800CW can be used to image tumor necrosis in vitro and in vivo. Further research will elucidate the application of [111In]In-DOTA-PEG4-800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors.

New ways to image and target tumour hypoxia and its molecular responses.

Tumour hypoxia and its molecular responses have been shown to be associated with poor prognosis. Detection of hypoxia, preferably in a non-invasive manner, could therefore predict treatment outcome and serve as a tool to individualize treatment. This review gives an overview of recent literature on hypoxia imaging markers currently used in clinical trials. Furthermore, recent progress made in targeting hypoxia (hypoxia-activated prodrugs) or hypoxia response (carbonic anhydrase IX inhibitors) is summarized. Last, window-of-opportunity trials implementing non-invasive imaging are proposed as an important tool to prove anti-tumour efficacy of experimental drugs early during drug development.