A novel EGFR inhibitor acts as potent tool for hypoxia-activated prodrug systems and exerts strong synergistic activity with VEGFR inhibition in vitro and in vivo.

Small-molecule EGFR inhibitors have distinctly improved the overall survival especially in EGFR-mutated lung cancer. However, their use is often limited by severe adverse effects and rapid resistance development. To overcome these limitations, a hypoxia-activatable Co(III)-based prodrug (KP2334) was recently synthesized releasing the new EGFR inhibitor KP2187 in a highly tumor-specific manner only in hypoxic areas of the tumor. However, the chemical modifications in KP2187 necessary for cobalt chelation could potentially interfere with its EGFR-binding ability. Consequently, in this study, the biological activity and EGFR inhibition potential of KP2187 was compared to clinically approved EGFR inhibitors. In general, the activity as well as EGFR binding (shown in docking studies) was very similar to erlotinib and gefitinib (while other EGFR-inhibitory drugs behaved different) indicating no interference of the chelating moiety with the EGFR binding. Moreover, KP2187 significantly inhibited cancer cell proliferation as well as EGFR pathway activation in vitro and in vivo. Finally, KP2187 proved to be highly synergistic with VEGFR inhibitors such as sunitinib. This indicates that KP2187-releasing hypoxia-activated prodrug systems are promising candidates to overcome the clinically observed enhanced toxicity of EGFR-VEGFR inhibitor combination therapies.

Prolonged Exposure to Oxaliplatin during HIPEC Improves Effectiveness in a Preclinical Micrometastasis Model.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients' samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Systematic Analysis of the Transcriptome Profiles and Co-Expression Networks of Tumour Endothelial Cells Identifies Several Tumour-Associated Modules and Potential Therapeutic Targets in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related death, with tumour associated liver endothelial cells being thought to be major drivers in HCC progression. This study aims to compare the gene expression profiles of tumour endothelial cells from the liver with endothelial cells from non-tumour liver tissue, to identify perturbed biologic functions, co-expression modules, and potentially drugable hub genes that could give rise to novel therapeutic targets and strategies. Gene Set Variation Analysis (GSVA) showed that cell growth-related pathways were upregulated, whereas apoptosis induction, immune and inflammatory-related pathways were downregulated in tumour endothelial cells. Weighted Gene Co-expression Network Analysis (WGCNA) identified several modules strongly associated to tumour endothelial cells or angiogenic activated endothelial cells with high endoglin (ENG) expression. In tumour cells, upregulated modules were associated with cell growth, cell proliferation, and DNA-replication, whereas downregulated modules were involved in immune functions, particularly complement activation. In ENG+ cells, upregulated modules were associated with cell adhesion and endothelial functions. One downregulated module was associated with immune system-related functions. Querying the STRING database revealed known functional-interaction networks underlying the modules. Several possible hub genes were identified, of which some (for example FEN1, BIRC5, NEK2, CDKN3, and TTK) are potentially druggable as determined by querying the Drug Gene Interaction database. In summary, our study provides a detailed picture of the transcriptomic differences between tumour and non-tumour endothelium in the liver on a co-expression network level, indicates several potential therapeutic targets and presents an analysis workflow that can be easily adapted to other projects.

The assessment of platelet function by thromboelastometry as a point-of-care test to guide Intercept-treated platelet support in hemato-oncological patients and hematopoietic stem cell transplantation recipients.

BACKGROUND: Pathogen inactivation (PI) techniques for platelet concentrates (PCs) are one of the latest innovations to improve blood safety and reduce the risk of transfusion-transmitted infections (TTIs). An impaired function and in vivo recovery of platelets as well as an increased PC demand are concerns regarding these techniques. The intent of this study was to evaluate the hemostatic effect of PCs treated with the Intercept™ System by thromboelastometry (TEM) and to assess the clinical validity of its results in comparison to post-transfusion increase (PTI) and corrected count increment (CCI). STUDY-DESIGN AND METHODS: This prospective-observational study included 47 patients (m:f = 25:22; median age: 54 years [21-70]) of our Bone Marrow Transplantation unit with hemato-oncological malignancies transfused with Intercept™-treated PCs. Serial TEM measurements were performed just before and 1 hour after PC transfusion and were analyzed for their correlation with PTI and CCI as well as for clinical variables. RESULTS: The majority of our patients had received a hematopoietic stem cell transplantation (HSCT) (n = 41; 87%). In median 9 (1-50) PCs were transfused. Serial TEM, PTI, and CCI measurements were available for 150 transfusion episodes. The median platelet dose transfused was 2.65 × 1011 /unit (1.8-6). The median CCI was 9.250 (0-28.000). We observed a significant improvement in TEM parameters (p < 0.05) after transfusion of PI PCs, which did not mandatory correlate with the 1-hour PTI and CCI. CONCLUSION: Serial TEM measurements indicate the hemostatic effect of Intercept™-treated PCs. The 1-hour PTI and CCI may not appropriately reflect the in vivo function of platelets after PI PC transfusion.

Systematic Review of Variations in Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for Peritoneal Metastasis from Colorectal Cancer.

BACKGROUND: Cytoreductive surgery (CRS), followed by hyperthermic intraperitoneal chemotherapy (HIPEC), combines radical surgery with abdominal heated chemotherapy, constituting a multimodal treatment approach. Since clear standards for HIPEC conduct in colorectal carcinoma (CRC) are lacking, we aimed to provide a comprehensive structured survey. Data sources and study eligibility criteria: A systematic literature search was performed in PubMed, with keywords "HIPEC" and "colorectal cancer", according to established guidelines. Articles were systematically screened, selecting 87 publications complemented by 48 publications identified through extended search for subsequent synthesis and evaluation, extracting inter alia details on used drugs, dosage, temperature, exposure times, and carrier solutions. RESULTS: Compiled publications contained 171 reports on HIPEC conduct foremost with mitomycin C and oxaliplatin, but also other drugs and drug combinations, comprising at least 60 different procedures. We hence provide an overview of interconnections between HIPEC protocols, used drugs and carrier solutions as well as their volumes. In addition, HIPEC temperatures and dosing benchmarks, as well as an estimate of in vivo resulting drug concentrations are demonstrated. CONCLUSIONS AND IMPLICATIONS: Owing to recent developments, HIPEC conduct and practices need to be reassessed. Unfortunately, imprecise and lacking reporting is frequent, which is why minimal information requirements should be established for HIPEC and the introduction of final drug concentrations for comparability reasons seems sensible.

Pharmacodynamics of Oxaliplatin-Derived Platinum Compounds During Hyperthermic Intraperitoneal Chemotherapy (HIPEC): An Emerging Aspect Supporting the Rational Design of Treatment Protocols.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is used to treat peritoneal surface malignancies with application of cytostatic drugs such as oxaliplatin (OX) after cytoreductive surgery. Despite its increased use, evidence for optimal drug dosage, and notably duration of HIPEC, is scarce. METHODS: In this study, OX distribution was comprehensively assessed in nine patients during HIPEC (300 mg OX/m2 body surface area in Physioneal solution for 30 min). Oxaliplatin and its derivatives were measured in peritoneal perfusates over time by liquid chromatography coupled with mass spectrometry (LC-MS), and the resulting total platinum concentration in tissue was analyzed by atomic absorption spectrometry. Additionally, a novel impedance-based real-time cytotoxicity assay was used to evaluate the bioactivity of perfusates ex vivo. RESULTS: Compared with amounts of OX expected in peritoneal perfusates by calculation, only 10-15% of the parent drug could be detected by LC-MS during HIPEC. Notably, the study additionally detected platinum compounds consistent with OX transformation, accounting for a further fraction of the applied drug. The cytotoxic properties of perfusates remained unchanged during HIPEC, with only a slight but significant attenuation evidenced after 30 min. CONCLUSIONS: The bioactivity of peritoneal perfusates ex vivo is a useful parameter for evaluating the actual cytotoxic potential of OX and its derivatives used in HIPEC over time, overcoming important limitations and disadvantages associated with respective drug monitoring only. Ex vivo cytotoxicity assays may be a promising tool to aid guiding future standardization and harmonization of HIPEC protocols based on drug-mediated effects.

Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

Dual non-responsiveness to antiplatelet treatment is a stronger predictor of cardiac adverse events than isolated non-responsiveness to clopidogrel or aspirin.

BACKGROUND: High platelet reactivity (HPR) under treatment with clopidogrel or aspirin is associated with adverse outcome. We aimed to investigate whether high platelet reactivity (HPR) to both aspirin and clopidogrel is a stronger predictor of adverse events compared to isolated HPR to clopidogrel or aspirin. METHODS: In this prospective cohort study platelet reactivity to adenosine diphosphate (ADP) and arachidonic acid (AA) was assessed by Multiple Electrode Aggregometry (MEA) in 403 patients undergoing percutaneous coronary intervention. The rates of the composite of cardiac adverse events (acute coronary syndrome, stent thrombosis, stroke, death and revascularization) were recorded during 12-month follow-up. RESULTS: The composite endpoint of cardiovascular adverse events occurred more often in patients with high platelet reactivity (HPR) to both agonists ADP and AA (37.5%) than in those with isolated HPR to ADP (33.3%), AA (25.6%) or without any HPR (18.6%; p=0.003). Classification tree analysis indicated that any HPR emerged as an independent predictor influencing outcome, which was associated with a 1.75 higher risk of cardiac adverse events (OR=1.75: 95%CI=1.1-2.9). Interestingly, the predictive value of HPR tended to be greater among patients with diabetes mellitus (OR=2.18; 95%CI=1.20-3.95). C-reactive protein and diabetes mellitus were independent predictors of high platelet reactivity to both agonists. CONCLUSIONS: Dual low responsiveness to clopidogrel and aspirin is a strong predictor of cardiac adverse events, especially in patients with diabetes mellitus, which underlines the need for personalized antiplatelet treatment.