Cell-Free Tumor DNA Detection-Based Liquid Biopsy of Plasma and Bile in Patients with Various Pancreatic Neoplasms.

The key challenge of cell-free tumor DNA (cftDNA) analysis in pancreatic ductal adenocarcinoma (PDAC) is overcoming its low detection rate, which is mainly explained by the overall scarcity of this biomarker in plasma. Obstructive jaundice is a frequent event in PDAC, which enables bile collection as a part of routine treatment. The aim of this study was to evaluate the performance of KRAS-mutated cftDNA detection-based liquid biopsy of plasma and bile in patients with pancreatic neoplasms using digital droplet PCR. The study included healthy volunteers (n = 38), patients with PDAC (n = 95, of which 20 had obstructive jaundice) and other pancreatic neoplasms (OPN) (n = 18). The sensitivity and specificity compared to the control group were 61% and 100% (AUC-ROC-0.805), and compared to the OPN group, they were 61% and 94% (AUC-ROC-0.794), respectively. Bile exhibited higher cftDNA levels than plasma (248.6 [6.743; 1068] vs. 3.26 [0; 19.225] copies/mL) and a two-fold higher detection rate (p < 0.01). Plasma cftDNA levels were associated with distant metastases, tumor size, and CA 19-9 (p < 0.05). The probability of survival was worse in patients with higher levels of cftDNA in plasma (hazard ratio-2.4; 95% CI: 1.3-4.6; p = 0.005) but not in bile (p > 0.05). Bile is a promising alternative to plasma in patients with obstructive jaundice, at least for the diagnostic purposes of liquid biopsy.

Microbiological and Cytokine Profiling of Menstrual Blood for the Assessment of Endometrial Receptivity: A Pilot Study.

Endometrial receptivity (ER) is a key factor required for the successful implantation of the embryo. However, the evaluation of ER is challenging, as a nondisruptive sampling of endometrial biomaterial by conventional methods is only possible outside of the embryo transfer (ET) cycle. We propose a novel approach for the assessment of ER-microbiological and cytokine profiling of menstrual blood aspirated directly from the uterine cavity at the beginning of the cryo-ET cycle. The aim of the pilot study was to evaluate its prognostic potential regarding the outcome of the in vitro fertilization procedure. Samples collected from a cohort of 42 patients undergoing cryo-ET were analyzed by a multiplex immunoassay (48 various cytokines, chemokines, and growth factors) and a real-time PCR assay (28 relevant microbial taxa and 3 members of the Herpesviridae family). Significant differences between groups of patients who achieved and did not achieve pregnancy were observed for G-CSF, GRO-α, IL-6, IL-9, MCP-1, M-CSF, SDF-1α, TNF-ß, TRAIL, SCF, IP-10, and MIG (p < 0.05), whereas microbial profiles were not associated with the outcome of cryo-ET. It appeared that levels of IP-10 and SCGF-ß were significantly lower (p < 0.05), in patients with endometriosis. Menstrual blood may provide great opportunities to noninvasively investigate various parameters of the endometrium.

Comparison of microbial profiles and viral status along the vagina-cervix-endometrium continuum of infertile patients.

For decades, the endometrium was considered to be a sterile environment. However, now this concept is disputed, and there is growing evidence that microbiota composition might affect endometrial receptivity. Routine clinical management of infertility is still limited to a microbiological assessment of the lower reproductive tract. The purpose of this study was to compare the abundance of various bacterial, fungal, and viral species, qualitatively and quantitatively, in vaginal, cervical, and endometrial biomaterial of infertile patients. A total of 300 samples from 100 infertile patients of a private assisted reproduction clinic were analyzed. A broad real-time polymerase chain reaction panel was used to identify 28 relevant microbial taxa as well as three members of the Herpesviridae family. All patients underwent endometrial biopsy for further histopathological evaluation. Analysis of the microbial diversity (within the boundaries of the detection panel) revealed that Shannon indexes in the cervix and vagina were similar (1.4 × 10-2 (1.6 × 10-3 - 6.5 × 10-1) vs 1.9 × 10-2 (2.3 × 10-3 - 5.3 × 10-1), respectively, p = 0.502), whereas endometrial indexes differed significantly from both regions (0 (0 - 1.4 × 10-1), p < 0.0001). Surprisingly, 17 microbial and viral taxa were detected in at least one sample. Endometrium exhibited a quite distinct microbiological profile, being different at the detection rates of 14 taxa (p < 0.05). Remarkably, 4% and 2% of endometrial samples were positive for Cytomegalovirus and Candida spp., respectively, while these were undetectable in corresponding cervical and vaginal samples. Prevalence of the Gardnerella vaginalis + Prevotella bivia + Porphyromonas spp. group in endometrium was associated with a low abundance of Lactobacillus spp. (p = 0.039). No noteworthy associations were identified between various microbiota characteristics and clinical parameters, such as chronic endometritis, uterine polyps and adhesions, endometriosis, and a history of sexually transmitted infections. These findings indicate that the microbiological profile of the endometrium is unique, and the analysis of the lower reproductive tract should supplement, rather than be a substitute for it.

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Blood ACE Phenotyping for Personalized Medicine: Revelation of Patients with Conformationally Altered ACE.

Background: The angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated blood ACE is a marker for granulomatous diseases and elevated ACE expression in tissues is associated with increased risk of cardiovascular diseases. Objective and Methodology: We applied a novel approach -ACE phenotyping-to find a reason for conformationally impaired ACE in the blood of one particular donor. Similar conformationally altered ACEs were detected previously in 2-4% of the healthy population and in up to 20% of patients with uremia, and were characterized by significant increase in the rate of angiotensin I hydrolysis. Principal findings: This donor has (1) significantly increased level of endogenous ACE inhibitor in plasma with MW less than 1000; (2) increased activity toward angiotensin I; (3) M71V mutation in ABCG2 (membrane transporter for more than 200 compounds, including bilirubin). We hypothesize that this patient may also have the decreased level of free bilirubin in plasma, which normally binds to the N domain of ACE. Analysis of the local conformation of ACE in plasma of patients with Gilbert and Crigler-Najjar syndromes allowed us to speculate that binding of mAbs 1G12 and 6A12 to plasma ACE could be a natural sensor for estimation of free bilirubin level in plasma. Totally, 235 human plasma/sera samples were screened for conformational changes in soluble ACE. Conclusions/Significance: ACE phenotyping of plasma samples allows us to identify individuals with conformationally altered ACE. This type of screening has clinical significance because this conformationally altered ACE could not only result in the enhancement of the level of angiotensin II but could also serve as an indicator of free bilirubin levels.

Mucosal biomarkers for endometrial receptivity: A promising yet underexplored aspect of reproductive medicine.

Annually, approximately 2 million assisted reproductive technology (ART) procedures are performed worldwide, of which, only ~25% lead to successful delivery. There are two major factors contributing to successful implantation: embryo quality and endometrial receptivity (ER). Although embryo quality might be assessed through morphological and genetic testing, no clinically approved techniques are available to evaluate ER. Mucus in different parts of the female reproductive tract contains many cytokines, chemokines, growth factors, and nucleic acids, which influence and reflect various implantation-related processes. Therefore, the aim of the present review was to summarize available data regarding noninvasively obtained mucosal biomarkers for ER and to investigate their ability to predict the outcome of ART procedures. A broad literature search was performed to define studies related to noninvasive ER assessments. More than 50 biomarkers detectable in endometrial fluid, embryo transfer cannula leftover cells and mucus, menstrual blood, cervicovaginal washings are discussed herein. The remarkable methodological heterogeneity of the reviewed studies complicates the comparison of their results. Nevertheless, certain promising analytical targets may already be identified, such as urocortin, activin A, IL-1ß, TNF-α, IP-10, MCP-1, and several oxidative stress biomarkers. The present review contains a collection of currently available mucosal biomarker-related data, which may provide insights for future studies.Abbreviations: ART: assisted reproductive technology; ER: endometrial receptivity; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; IUI: intrauterine insemination; MeSH: Medical Subject Headings; hDP 200: human decidua-associated protein 200; ET: embryo transfer; IL-18: Interleukin-18; LRG: leucine-rich α2-glycoprotein; ROC: receiver operating characteristic; AUC: area under the ROC-curve; LH: luteinizing hormone; LIF: leukemia inhibitory factor; TNF-α: tumor necrosis factor alpha; IFN-γ: interferon γ; MCP-1: monocyte chemoattractant protein-1; VEGF: vascular endothelial growth factor; SOD: superoxide dismutase; CAT: catalase; LPO: lipid peroxidation; TTG: total thiol groups; TAP: total antioxidant power; CE: chronic endometritis.

Urine TERT promoter mutations-based tumor DNA detection in patients with bladder cancer: A pilot study.

Telomerase reverse transcriptase (TERT) promoter mutations are the most frequent genetic events in bladder cancer (BC). The aim of the present pilot study was to evaluate the diagnostic potential of urine TERT promoter mutations-based liquid biopsy in patients with an ongoing oncological process, as well as in post-resection patients at risk of BC recurrence. A total of 60 patients were enrolled, of whom 27 patients had histologically proven BC; 23 had no signs of BC (control group); and 10 patients underwent transurethral malignancy resection 3-6 months prior to urine donation ('second look' group). Urine TERT promoter mutations were detected using Droplet Digital PCR. Receiver operating characteristic curve analysis revealed significant diagnostic power of the present approach (area under the curve: -0.768). At the cut-off value of tumor DNA fraction 0.34%, the sensitivity and specificity were 55.56 and 100%, respectively. In the positive samples, tumor DNA fraction varied significantly from 0.59 to 48.77%. In the 'second look' group, tumor DNA was detected in 4/10 patients, highlighting the possibility of BC recurrence with its fraction ranging only from 0.90 to 6.61%. Therefore, urine TERT promoter mutations-based liquid biopsy appears to be a promising tool for BC diagnosis and surveillance. The main study will include recruitment of additional patients, extension of the mutation panel, prolonged follow-up of the post-resection patients, as well as screening of industrial workers exposed to specific carcinogens.

Phenotyping Angiotensin-Converting Enzyme in Blood: A Necessary Approach for Precision Medicine.

BACKGROUND: Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by ACE in blood) is associated with increased risk of cardiovascular diseases. Elevated ACE in blood is also a marker for granulomatous diseases. METHODS: We applied our novel approach-ACE phenotyping-to characterize serum ACE in 300 unrelated patients and to establish normal values for ACE levels. ACE phenotyping includes (a) determination of ACE activity with 2 substrates (Z-Phe-His-Leu [ZPHL] and Hip-His-Leu [HHL]), (b) calculation of a ratio for hydrolysis of ZPHL and HHL, and (c) quantification of ACE immunoreactive protein levels and ACE conformation with a set of monoclonal antibodies (mAbs) to ACE. RESULTS: Only a combination of ACE activity determination with 2 substrates and quantification of the amount of ACE immunoreactive protein with mAbs 1G12 and 9B9 allows for the unequivocal detection of the presence of ACE inhibitors in the blood. After excluding such subjects, we were able to establish normal values of ACE in healthy populations: 50%-150% from control pooled serum. This ACE phenotyping approach in screening format with special attention to outliers can also identify patients with various mutations in ACE and may help to identify the as yet unknown ACE secretase or other mechanistic details of precise regulation of ACE expression. CONCLUSIONS: ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques by establishing different risk groups based on ACE phenotype.

Novel ACE mutations mimicking sarcoidosis by increasing blood ACE levels.

An elevated blood angiotensin I-converting enzyme (ACE) supports diagnosis of sarcoidosis and Gaucher disease. However, some ACE mutations increase ACE shedding, and patients with these mutations are therefore at risk of being incorrectly diagnosed with sarcoidosis because of elevated serum ACE levels. We applied a novel approach called "ACE phenotyping" to identify possible ACE mutations in 3 pulmonary clinic patients that had suspected sarcoidosis based on elevated blood ACE levels. Conformational fingerprinting of ACE indicated that these mutations may be localized in the stalk region of the protein and these were confirmed by whole exome sequencing. Index patient 1 (IP1) had a mutation (P1199L) that had been previously identified, while the other 2 patients had novel ACE mutations. IP2 had 2 mutations, T887M and N1196K (eliminating a putative glycosylation site), while IP3 had a stop codon mutation Q1124X (eliminating the transmembrane anchor). We also performed a comprehensive analysis of the existing database of all ACE mutations to estimate the proportion of mutations increasing ACE shedding. The frequency of ACE mutations resulting in increased blood ACE levels may be much higher than previously estimated. ACE phenotyping, together with whole exome sequencing, is a diagnostic approach that could prevent unnecessary invasive and/or costly diagnostic procedures, or potentially harmful treatment for patients misdiagnosed on the basis of elevated blood ACE levels.

Direct comparison of QIAamp DSP Virus Kit and QIAamp Circulating Nucleic Acid Kit regarding cell-free fetal DNA isolation from maternal peripheral blood.

BACKGROUND: Blood of pregnant women contains cell-free fetal DNA (cffDNA), which is widely used in non-invasive prenatal diagnosis. The modern laboratory equipment market provides huge variety of commercial kits for isolation of circulating nucleic acids, but unfortunately none of them are standardized for isolation of cffDNA, which is a crucial step for success of subsequent analysis. AIM: To compare DSPVK and CNAK in terms of cffDNA, cell-free total DNA (cftDNA) yield and resulting cffDNA fraction, as well as to try to explain the possible difference between the efficacy of these kits. METHODS: Peripheral blood samples were collected from 18 healthy pregnant women (6th-14th week of pregnancy) and from 12 healthy unpregnant subjects. cftDNA was isolated using QIAamp Circulating Nucleic Acid Kit (CNAK) (Qiagen, Germany) and QIAamp DSP Virus Kit (DSPVK) (Qiagen, Germany) from 1 ml of plasma of each sample. Methylation-sensitive restriction was carried out to isolate cffDNA. Yield of cffDNA and cftDNA was quantified using digital PCR. To explain the difference in resulting efficacy of these two kits PCR inhibitors analysis was performed, as well as the optimal plasma input for DSPVK was investigated. RESULTS: Yield of cffDNA using CNAK was statistically significantly higher than using DSPVK (167.62 (125.34-192.47) vs 52.88 (35.48-125.42) GEq/mL, p < 0.001). The same applies to cftDNA yield, CNAK appears to be statistically significantly superior to DSPVK (743.42 (455.02-898.33) vs 371.07 (294.37-509.89) GEq/mL, p < 0.001). cffDNA fraction using CNAK was also higher than using DSVPK (24.75 (14.5-31.53) vs 14.20 (6.88-25.83) %, p = 0.586), although the difference was not statistically significant due to inconsistency of DSPVK results from sample to sample. PCR inhibitors analysis uncovered increased amount of PCR inhibitors in CNAK cftDNA solution, compared to DSPVK (p = 0.002). Usage of 0.5 mL of plasma for cftDNA extraction with DSPVK over 1 mL demonstrates almost 1.8 times higher cftDNA output (p = 0.028), which suggests that this kit is not so viable for volumes of plasma larger than 0.5 mL. CONCLUSIONS: We recommend CNAK over DSPVK for quantitative analysis of cffDNA. Nevertheless, DSPVK is definitely suitable for qualitative analysis as well as for research with limited budget, since it is almost 3 times cheaper than CNAK.