NormiRazor: tool applying GPU-accelerated computing for determination of internal references in microRNA transcription studies.

BACKGROUND: Multi-gene expression assays are an attractive tool in revealing complex regulatory mechanisms in living organisms. Normalization is an indispensable step of data analysis in all those studies, since it removes unwanted, non-biological variability from data. In targeted qPCR assays it is typically performed with respect to prespecified reference genes, but the lack of robust strategy of their selection is reported in literature, especially in studies concerning circulating microRNAs (miRNA). Unfortunately, this problem impedes translation of scientific discoveries on miRNA biomarkers into widely available laboratory assays. Previous studies concluded that averaged expressions of multi-miRNA combinations are more stable references than single genes. However, due to the number of such combinations the computational load is considerable and may be hindering for objective reference selection in large datasets. Existing implementations of normalization algorithms (geNorm, NormFinder and BestKeeper) have poor performance and may require days to compute stability values for all potential reference as the evaluation is performed sequentially. RESULTS: We designed NormiRazor - an integrative tool which implements those methods in a parallel manner on a graphics processing unit (GPU) using CUDA platform. We tested our approach on publicly available miRNA expression datasets. As a result, the times of executions on 8 datasets containing from 50 to 400 miRNAs (subsets of GSE68314) decreased 18.7 ±0.6 (mean ±SD), 104.7 ±4.2 and 76.5 ±2.2 times for geNorm, BestKeeper and NormFinder with respect to previous Python implementation. To allow for easy access to normalization pipeline for biomedical researchers we implemented NormiRazor as an online platform where a user could normalize their datasets based on the automatically selected references. It is available at norm.btm.umed.pl, together with instruction manual and exemplary datasets. CONCLUSIONS: NormiRazor allows for an easy, informed choice of reference genes for qPCR transcriptomic studies. As such it can improve comparability and repeatability of experiments and in longer perspective help translate newly discovered biomarkers into readily available assays.

A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs.

BACKGROUND: The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach. RESULTS: We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets. CONCLUSIONS: No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.

Discrepancies between methods of continuous glucose monitoring in key metrics of glucose control in children with type 1 diabetes.

OBJECTIVE: We aimed to compare glycemic control and variability parameters obtained from paired records of real-time continuous glucose monitoring (RT-CGM) and flash glucose monitoring (FGM). METHODS: Ten Polish boys and 11 girls aged 15.3 ± 2.1 years with type 1 diabetes for 7.7 ± 4.5 years and glycated hemoglobin 7.35 ± 0.7% (57 ± 5 mmol/mol) were recruited between August 2017 and June 2018 and equipped with devices for RT-CGM (iPro2 system with Enlite electrodes) and FGM (FreeStyle Libre) for 1 week. Afterwards, Glyculator 2.0 software was used to calculate and compare key metrics of glycemic control listed in the International Consensus on Use of Continuous Glucose Monitoring, with distinction into all record/night-time/day-time blocks when appropriate. RESULTS: Agreement between the two systems' measurements across patients ranged from poor (R2 = .39) to nearly perfect (R2 = .97). Significant differences between RT-CGM and FGM were observed in five important metrics: coefficient of variation (median difference: -4.12% [25%-75%: -7.50% to -2.96%], P = .0001), low blood glucose index (-0.88 [-1.88 to -0.18], P = .0004), % of time below 70 mg/dL (3.9 mmol/L) (-4.77 [-8.39 to -1.19], P = .0015) and 54 mg/dL (3 mmol/L) (-1.33 [-4.07 to 0.00], P = .0033) and primary time in range (TIR) 70-180 mg/dL (8.58 [1.31 to 12.66], P = .0006). CONCLUSIONS: RT-CGM and FGM differ in their estimates of clinically important indices of glycemic control. Therefore, such metrics cannot be directly compared between people using different systems. Our result necessitates system-specific guidelines and targets if TIR and glycemic variability are to be used as an endpoint in clinical trials.

Does the presence of sentinel lymph node macrometastases in breast cancer patients require axillary lymph node dissection?-Single-center analysis.

According to the current guidelines on treatment of breast cancer patients, identification of metastases in the sentinel lymph node (SLN (+)) is not an absolute indication for necessary axillary lymph node dissection (ALND). In our study, we present long-term outcomes of treatment among SLN(+) patients referred for conservative treatment, for example, no further ALND. A total of 3145 breast cancer patients subjected to sentinel lymph node biopsy (SLNB) between November 2008 and June 2015. SLN metastases were identified in 719 patients (22.9%). Locoregional recurrences and distant metastases as endpoints were distinquished. The mean follow-up time for patients after ALND was 36.2 months (6-74 months); 18.8 months (6-38 months) for patients with SLN macrometastases without ALND; and 34.0 months (6-74 months) for patients with micrometastases. Adjuvant ALND was performed in 626 of SLN(+) patients. Conservative treatment was applied in the remaining 93 cases. Among SLN(+) patients without adjuvant ALND, there was one case of recurrence (1.07%). In the group of patients without SLN, metastases recurrence was noted in 32 patients (1.32%). Among SLN(+) patients diagnosed with macrometastases, recurrence concerned 2.01% of analyzed cases (all subjected to ALND). Lack of radical surgical treatment in SLN(+) breast cancer patients did not lead to worsening long-term outcomes. In the occurrence of macrometastases to the sentinel lymph node, abandoning completion axillary lymph node dissection might be a reasonable option. However, it would require continuation of current research, preferably involving a clinical trial.

Polo-like kinase-1 immunoreactivity is associated with metastases in cutaneous melanoma.

BACKGROUND: Polo-like kinase-1 (PLK-1) is one of the key regulators of cell cycle progression. Increased expression of PLK-1 was observed in several tumor types. METHODS: We immunohistochemically assessed PLK-1 expression in neoplastic and stromal compartments of 96 cutaneous melanomas, and analyzed associations between PLK-1 expression and clinicopathological characteristics. RESULTS: PLK-1 expression in cancer cells was not associated with basic clinical (eg, age, gender and tumor location) or histopathological (eg, Breslow thickness, mitotic rate and ulceration) parameters. However, increased PLK-1 was more frequent in tumors with concurrent regional nodal metastases and positive sentinel lymph node biopsy status. All primary tumors associated with co-existing distant metastases exhibited high PLK-1 expression in melanoma cells. Conversely, PLK-1 expression in stromal cells was more frequent in tumors without nodal metastases. PLK-1 expression in both compartments was not associated with survival. CONCLUSION: PLK-1 expression is associated with metastatic potential in cutaneous melanoma.

A Translational Model to Improve Early Detection of Epithelial Ovarian Cancers.

Neural network analyses of circulating miRNAs have shown potential as non-invasive screening tests for ovarian cancer. A clinically useful test would detect occult disease when complete cytoreduction is most feasible. Here we used murine xenografts to sensitize a neural network model to detect low volume disease and applied the model to sera from 75 early-stage ovarian cancer cases age-matched to 200 benign adnexal masses or healthy controls. The 14-miRNA model efficiently discriminated tumor bearing animals from controls with 100% sensitivity down to tumor inoculums of 50,000 cells. Among early-stage patient samples, the model performed well with 73% sensitivity at 91% specificity. Applied to a population with 1% disease prevalence, we hypothesize the model would detect most early-stage ovarian cancers while maintaining a negative predictive value of 99.97% (95% CI 99.95%-99.98%). Overall, this supports the concept that miRNAs may be useful as screening markers for early-stage disease.

Analysis of the impact of bronchial asthma and hypersensitivity to aspirin on the clinical course of chronic sinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a disease with still not enough known pathogenesis despite the development of genetics, immunological and microbiological research. The number of patients with CRS has been constantly growing. The coexistence of CRS, bronchial asthma and aspirin intolerance (aspirin triad) is an adverse prognostic factor with higher risk of recurrences. The aim of study was to compare the severity of CRSwNP depending of coexistence of bronchial asthma and/or aspirin intolerance. The research was performed in the group of 204 patients operated 2009-2013 with 5 years follow-up. Higher nasal polyps growth in groups of patients with aspirin triad and CRSwNP and bronchial asthma in endoscopic examination (p=0,0005 and p=0,0030 respectively) and CT-scan according to Lund-Mackay point scale (p<0,0001 and p=0,0009) was showed. Also, these patients presented increased severity of nasal symptoms before surgical treatment according to VAS scale (p=0,0126 for CRSwNP with bronchial asthma; p=0,0390 for aspirin triad). Similarly, 6 months after surgery the same groups of patients presented higher severity of the disease symptoms (p<0,0001 for aspirin triad' patients; p=0,0174 for CRSwNP and bronchial asthma' patients) . Patients with aspirin triad had also statistically more surgeries in past (p=0,001), what proves that recurrences in this group are very likely to be observed in spite of the use of proper conservative treatment. No such differences have been shown in the group of patients with CRSwNP and isolated aspirin intolerance (without bronchial asthma). Allergy to inhaled allergens, hypersensitivity to aspirin are factors significantly worsening the course of CRSwNP. It would be advisable to consider, despite a lack of history of aspirin intolerance, a hypersensititvity to aspirin test in patients with particularly severe CRSwNP, especially those associated with bronchial asthma. It also seems reasonable to carry out such a test on every patient with newly diagnosed CRSwNP and bronchial asthma in order to be able to plan further treatment in this group of patients accordingly including biological treatment with antimonoclonal therapy against interleukin 4, 5 or13.

Resistance to Data Loss of Glycemic Variability Measurements in Long-Term Continuous Glucose Monitoring.

Background: Continuous glucose monitoring (CGM) is a method of estimating blood glucose values from those recorded in the interstitial fluid. Because increasingly longer CGM measurements are possible, errors and data loss become more and more likely and potentially more damaging to accurate calculations of glycemic variability (GV) indices. Our research investigates the resistance of the CGM recording to data loss. Methods: We collected 71 CGM recordings (duration of min: 2, max: 265, median: 42 days) from patients with type 1 diabetes and used three algorithms to introduce missing data. We calculated mean and standard deviation (SD) of absolute percentage error of 12 variability indices and correlated those with the percentage of missing data and duration of the measurements. Results: Mean absolute percentage error of variability indices increased linearly with the percentage of missing data along with SD of absolute percentage error. Except for mean amplitude of glycemic excursions and time spent in hypoglycemia, all absolute errors never exceeded 25%, while mean absolute errors stayed below 5%. The gradient removal algorithm introduced errors larger than the single datapoint and block removal algorithms. The absolute percentage error of variability indices correlated negatively with the duration of the CGM measurements. Conclusions: Standard GV measurements in long-term glucose monitoring are robustly resistant to data loss.

Upregulation of FOXP1 is a new independent unfavorable prognosticator and a specific predictor of lymphatic dissemination in cutaneous melanoma patients.

BACKGROUND: FOXP1 is a pleiotropic protein that plays important roles in immune responses (B-cell development regulation and differentiation of monocyte), organ development (cardiac valves, lung, and esophagus), and neuronal development. Besides being the primary regulator of normal human tissue development, FOXP1 also plays a role in tumorigenesis. However, the potential value of FOXP1 expression in tumor prognosis remains controversial. FOXP1 expression was assessed in tumor cells (TCs) and stromal cells (SCs) of cutaneous melanomas with the aim of analyzing the associations between FOXP1 expression and clinicopathological characteristics. We believe this article to be the first report analyzing the correlations between FOXP1 expression and clinicopathological, as well as histological, characteristics in melanoma. MATERIALS AND METHODS: In total, 96 formalin-fixed, paraffin-embedded primary cutaneous melanoma tissue specimens were subjected to immunohistochemical analysis for FOXP1, and the results were correlated with classical clinicopathological features and patient survival. RESULTS: FOXP1 overexpression in TCs was strongly associated with the presence of metastases in sentinel lymph nodes (p=0.0003, OR=11.66) and positive status of regional lymph nodes (p=0.0006, OR=22.15). In 96% (52 of 54) of patients presenting with low FOXP1 expression, no clinical or histopathological features of lymphatic dissemination were observed. However, thinner and nonulcerated tumors were reported to have increased numbers of FOXP1-positive SCs. In addition, a strong association was observed between FOXP1 upregulation in SCs and the absence of regional lymph node metastases. There was a significant correlation between FOXP1 upregulation in TCs and shorter cancer-specific overall survival (log-rank test, p=0.0040) and disease-free survival (log-rank test, p=0.0021). FOXP1 expression was confirmed in multivariate analysis as a factor that significantly unfavorably impacts prognosis in melanoma patients (HR=3.14, p=0.0299, adjusted for age, Breslow thickness, and sex). CONCLUSION: The findings from this study indicate that FOXP1 has a major role in melanoma progression, which makes it a candidate for molecular target-based cancer therapy.