Immune checkpoints as potential theragnostic biomarkers for epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is the leading cause of death associated with gynecologic tumors. EOC is asymptomatic in early stages, so most patients are not diagnosed until late stages, highlighting the need to develop new diagnostic biomarkers. Mediators of the tumoral microenvironment may influence EOC progression and resistance to treatment. AIM: To analyze immune checkpoints to evaluate them as theranostic biomarkers for EOC. PATIENTS AND METHODS: Serum levels of 16 immune checkpoints were determined in EOC patients and healthy controls using the MILLIPLEX MAP® Human Immuno-Oncology Checkpoint Protein Magnetic Bead Panel. RESULTS: Seven receptors: BTLA, CD40, CD80/B7-1, GITRL, LAG-3, TIM-3, TLR-2 are differentially expressed between EOC and healthy controls. Serum levels of immune checkpoints in EOC patients are positively significantly correlated with levels of their ligands, with a higher significant correlation between CD80 and CTLA4 than between CD28 and CD80. Four receptors, CD40, HVEM, PD-1, and PD-L1, are positively associated with the development of resistance to Taxol-platinum-based chemotherapy. All of them have an acceptable area under the curve (>0.7). CONCLUSION: This study has yielded a first panel of seven immune checkpoints (BTLA, CD40, CD80/B7-1, GITRL, LAG-3, TIM-3, TLR-2) associated with a higher risk of EOC and a second panel of four immune checkpoints (CD40, HVEM, PD-1, PD-L1) that may help physicians to identify EOC patients who are at high risk of developing resistance to EOC chemotherapy.

A Magnetoelectrochemical Bioassay for Highly Sensitive Sensing of Point Mutations in Interleukin-6 Gene Using TMB as a Hybridization Intercalation Indicator.

Point mutations are common in the human DNA genome and are closely related to higher susceptibility to cancer diseases. Therefore, suitable methods for their sensing are of general interest. In this work, we report on a magnetic electrochemical bioassay using DNA probes tethered to streptavidin magnetic beads (strep-MBs) to detect T > G single nucleotide polymorphism (SNP) within the inteleukin-6 (IL6) gene in human genomic DNA. In the presence of the target DNA fragment and tetramethylbenzidine (TMB), the electrochemical signal related to the oxidation of TMB is observed, which is much higher than the one obtained in the absence of the target. The key parameters affecting the analytical signal, such as the concentration of the biotinylated probe, its incubation time with strep-MBs, DNA hybridization time, and TMB loading, were optimized using the electrochemical signal intensity and signal-to-blank (S/B) ratio as selection criteria. Using spiked buffer solutions, the bioassay can detect the mutated allele in a wide range of concentrations (over six decades) with a low detection limit (7.3 fM). Furthermore, the bioassay displays a high specificity with high concentrations of the major allele (one mismatched), and two mismatched and non-complementary DNA. More importantly, the bioassay can detect the variation in scarcely diluted human DNA, collected from 23 donors, and can reliably distinguish between heterozygous (TG genotype) and homozygous (GG genotype) in respect to the control subjects (TT genotype), where the differences are statistically highly significant (p-value < 0.001). Thus, the bioassay is useful for cohort studies targeting one or more mutations in human DNA.

Identification of two theranostic biomarker panels for epithelial ovarian cancer.

BACKGROUND: Epithelial Ovarian cancer (EOC) is the leading cause of death associated with gynecologic tumors. Because the disease is asymptomatic in early-stage, the majority of patients are not diagnosed until late stages, highlighting the need for the development of novel diagnostic biomarkers. Mediators of tumoral microenvironment may affect EOC progression and resistance to treatment. AIM OF THE STUDY: Analysis of serum proteins to identify a panel of theranostic biomarkers for EOC. PATIENTS AND METHODS: Serum levels of 65 analytes were determined in EOC patients, and healthy controls with the ProcartaPlex Human Immune Monitoring 65-Plex Panel. RESULTS: Twenty-one analytes: 7 cytokines (IFN-γ, IL-12p70, IL-13, IL-18 and TSLP), 7 chemokines (Eotaxin, eotaxin-2, IP-10, BLC, I-TAC, SDF-1α, and fractalkine), 2 growth factors (MMP-1, VEGF-α), and 5 soluble receptors (APRIL, CD40L, TWEAK, CD30 and TNFRII; were significantly differentially expressed between the two groups. ROC curves showed that only seven of them (IL-9, TNF-α, Eotaxin, IP-10, BLC, Fractalkine, and Tweak) had AUC values greater than 0.70 and thus had potential clinical utility. Moreover, five cytokines: IFN-γ, IL-1 ß, IL-8, MIP-1ß, and TNF-α are positively associated with patients who developed resistance to taxol-platinum-based chemotherapy (CT). CONCLUSION: This study has revealed a first panel of 7 analytes (IL-9, TNF-α, Eotaxin, IP-10, BLC, Fractalkine and Tweak) that can be used for early detection of EOC and a second panel of five cytokines (IFN-γ, IL-1ß, IL-8, MIP-1ß, TNF-α) that can help clinicians to identify EOC patients who are at higher risk to develop resistance to CT of EOC.

Sensitive electrochemical detection of polymorphisms in IL6 and TGFß1 genes from ovarian cancer DNA patients using EcoRI and DNA hairpin-modified gold electrodes.

Two electrochemical bioplatforms were prepared based on thiolated hairpin DNA probes tethered to AuNP-modified screen-printed electrodes to detect T > G and T > C polymorphisms, namely rs1880269 and rs1800469, present the interleukin-6 (IL6) and transforming growth factor ß1 (TGFß1) genes. The electrochemical readout was ensured by the detection of the double-stranded DNA using methylene blue as a redox probe after treatment by EcoRI restrictase. The main parameters influencing the analytical response such as the thiolated DNA probe concentration, incubation time with electrode, DNA hybridization time, EcoRI enzyme load, and its cleavage time were optimized based on the current intensity and signal-to-blank (S/B) ratio as selection criteria. Using spiked buffer solutions, the IL6 and TGFß1 E-bioplatforms display wide ranges of linearity (1 × 102-1 × 108 fM and 5 × 101-1 × 105 fM, respectively) and limits of detection (47.9 fM and 16.6 fM, respectively). The two bioelectrodes have also good discrimination toward 1-mismatched, two mismatched, and non-complementary sequences, when they were used 30-fold higher than the target sequences. More importantly, the two bioplatforms successfully detected the single nucleotide polymorphisms (SNPs) in scarcely diluted genomic DNA, collected from 52 donors, and showed they can reliably distinguish between heterozygous (TG and TC genotypes) and homozygous (GG and CC genotypes) patients with  respect to the control subjects (TT genotype), where the differences are statistically highly significant (p-value < 0.0001). Thus, the designed devices could be used to conduct large cohort studies targeting these mutations or extended to other SNPs.