Multiplexed serum biomarkers to discriminate nonviable and ectopic pregnancy.

OBJECTIVE: To evaluate combinations of candidate biomarkers to develop a multiplexed prediction model for identifying the viability and location of an early pregnancy. In this study, we assessed 24 biomarkers with multiple machine learning-based methodologies to assess if multiplexed biomarkers may improve the diagnosis of normal and abnormal early pregnancies. DESIGN: A nested case-control design evaluated the predictive ability and discrimination of biomarkers in patients at risk of early pregnancy failure in the first trimester to classify viability and location. SETTING: Three university hospitals. PATIENTS: A total of 218 individuals with pain and/or bleeding in early pregnancy: 75 had an ongoing intrauterine gestation; 68 had ectopic pregnancies (EPs); and 75 had miscarriages. INTERVENTIONS: Serum levels of 24 biomarkers were assessed in the same patients. Multiple machine learning-based methodologies to evaluate combinations of these top candidates to develop a multiplexed prediction model for the identification of a nonviable pregnancy (ongoing intrauterine pregnancy vs. miscarriage or EP) and an EP (EP vs. ongoing intrauterine pregnancy or miscarriage). MAIN OUTCOME MEASURES: The predicted classification using each model was compared with the actual diagnosis, and sensitivity, specificity, positive predictive value, negative predictive value, conclusive classification, and accuracy were calculated. RESULTS: Models using classification regression tree analysis using 3 (pregnancy-specific beta-1-glycoprotein 3 [PSG3], chorionic gonadotropin-alpha subunit, and pregnancy-associated plasma protein-A) biomarkers were able to predict a maximum sensitivity of 93.3% and a maximum specificity of 98.6%. The model with the highest accuracy was 97.4% (with 70.2% receiving classification). Models using an overlapping group of 3 (soluble fms-like tyrosine kinase-1, PSG3, and tissue factor pathway inhibitor 2) biomarkers achieved a maximum sensitivity of 98.5% and a maximum specificity of 95.3%. The model with the highest accuracy was 94.4% (with 65.6% receiving classification). When the models were used simultaneously, the conclusive classification increased to 72.7% with an accuracy of 95.9%. The predictive ability of the biomarkers in the random forest produced similar test characteristics when using 11 predictive biomarkers. CONCLUSION: We have demonstrated a pool of biomarkers from divergent biological pathways that can be used to classify individuals with potential early pregnancy loss. The biomarkers choriogonadotropin alpha, pregnancy-associated plasma protein-A, and PSG3 can be used to predict viability, and soluble fms-like tyrosine kinase-1, tissue factor pathway inhibitor 2, and PSG3 can be used to predict pregnancy location.

Prenatal screening for preeclampsia: the roles of placental growth factor and pregnancy-associated plasma protein A in the first trimester and placental growth factor and soluble fms-like tyrosine kinase 1-placental growth factor ratio in the early second trimester.

BACKGROUND: Professional societies have recommended universal first trimester screening for preeclampsia and a second or third trimester soluble fms-like tyrosine kinase-1-placental growth factor ratio test to assess for preeclampsia and its severity. However, it may not be feasible to implement the most optimal screening protocol for preeclampsia in the first trimester which uses a combination of maternal characteristics, maternal biophysical and biochemical markers due to limitations in the access to uterine artery doppler ultrasound. There are inconsistent findings on how early in the second trimester the fms-like tyrosine kinase-1-placental growth factor ratio begins to provide useful information in preeclampsia prediction. OBJECTIVE: This study aimed to assess the accuracy of (1) a combination of maternal characteristics, maternal serum pregnancy-associated plasma protein A, and placental growth factor in the screening for preeclampsia in the first trimester; and (2) placental growth factor or soluble fms-like tyrosine kinase-1-placental growth factor ratio in the prediction of preeclampsia in the early second trimester. STUDY DESIGN: This retrospective case-control study used frozen residual blood samples from women who had aneuploidy screening and delivered at a tertiary center. The case group included pregnancies with gestational hypertension or preeclampsia (further classified as early-onset [birth at <34 weeks' gestation] and preterm preeclampsia [birth at <37 weeks' gestation]). Each case was matched with 3 control pregnancies by date of blood sample draw, gestational age at first blood sample draw, maternal age, maternal ethnicity, type of multiple-marker screening, and amount of residual sample. Mann-Whitney U tests were used to assess the associations between serum markers and the risk of preeclampsia. Logistic regressions were used to assess if the risk of preeclampsia can be predicted using a combination of maternal characteristics and serum markers. RESULTS: The case group included 146 preeclampsia and 295 gestational hypertension cases. Compared with the controls, preeclampsia cases had significantly lower first-trimester pregnancy-associated plasma protein A and placental growth factor. At a 20% false-positive rate, 71% of early-onset and 58% of preterm preeclampsia cases can be predicted using maternal characteristics, pregnancy-associated plasma protein A, and placental growth factor. Preeclampsia cases had lower second-trimester placental growth factor and a higher soluble fms-like tyrosine kinase-1-placental growth factor ratio. At a 10% false-positive rate, 80% and 53% of early-onset preeclampsia can be predicted using maternal characteristics and placental growth factor or soluble fms-like tyrosine kinase-1-placental growth factor ratio, respectively. CONCLUSION: The current first-trimester aneuploidy screening programs may be expanded to identify women at increased risk of developing preeclampsia. Early in the second trimester, placental growth factor alone provided better prediction for preeclampsia compared with the soluble fms-like tyrosine kinase-1-placental growth factor ratio.

Modified multiple marker aneuploidy screening as a primary screening test for preeclampsia.

BACKGROUND: Abnormal levels of maternal biochemical markers used in multiple marker aneuploidy screening have been associated with adverse pregnancy outcomes. This study aims to assess if a combination of maternal characteristics and biochemical markers in the first and second trimesters can be used to screen for preeclampsia (PE). The secondary aim was to assess this combination in identifying pregnancies at risk for gestational hypertension and preterm birth. METHODS: This case-control study used information on maternal characteristics and residual blood samples from pregnant women who have undergone multiple marker aneuploidy screening. The median multiple of the median (MoM) of first and second trimester biochemical markers in cases (women with PE, gestational hypertension and preterm birth) and controls were compared. Biochemical markers included pregnancy-associated plasma protein A (PAPP-A), placental growth factor (PlGF), human chorionic gonadotropin (hCG), alpha feto-protein (AFP), unconjugated estriol (uE3) and Inhibin A. Logistic regression analysis was used to estimate screening performance using different marker combinations. Screening performance was defined as detection rate (DR) and false positive rate (FPR). Preterm and early-onset preeclampsia PE were defined as women with PE who delivered at < 37 and < 34 weeks of gestation, respectively. RESULTS: There were 147 pregnancies with PE (81 term, 49 preterm and 17 early-onset), 295 with gestational hypertension, and 166 preterm birth. Compared to controls, PE cases had significantly lower median MoM of PAPP-A (0.77 vs 1.10, p < 0.0001), PlGF (0.76 vs 1.01, p < 0.0001) and free-ß hCG (0.81 vs. 0.98, p < 0.001) in the first trimester along with PAPP-A (0.82 vs 0.99, p < 0.01) and PlGF (0.75 vs 1.02, p < 0.0001) in the second trimester. The lowest first trimester PAPP-A, PlGF and free ß-hCG were seen in those with preterm and early-onset PE. At a 20% FPR, 67% of preterm and 76% of early-onset PE cases can be predicted using a combination of maternal characteristics with PAPP-A and PlGF in the first trimester. The corresponding DR was 58% for gestational hypertension and 36% for preterm birth cases. CONCLUSIONS: Maternal characteristics with first trimester PAPP-A and PlGF measured for aneuploidy screening provided reasonable accuracy in identifying women at risk of developing early onset PE, allowing triage of high-risk women for further investigation and risk-reducing therapy. This combination was less accurate in predicting women who have gestational hypertension or preterm birth.

A phylogenetic overview of the Hydnaceae (Cantharellales, Basidiomycota) with new taxa from China.

The family Hydnaceae (Cantharellales, Basidiomycota) is a group of fungi found worldwide which exhibit stichic nuclear division. The group is highly diverse in morphology, ecology, and phylogeny, and includes some edible species which are popular all over the world. Traditionally, Hydnaceae together with Cantharellaceae, Clavulinaceae and Sistotremataceae are four families in the Cantharellales. The four families were combined and redefined as "Hydnaceae", however, a comprehensive phylogeny based on multiple-marker dataset for the entire Hydnaceae sensu stricto is still lacking and the delimitation is also unclear. We inferred Maximum Likelihood and Bayesian phylogenies for the family Hydnaceae from the data of five DNA regions: the large subunit of nuclear ribosomal RNA gene (nLSU), the internal transcribed spacer regions (ITS), the mitochondrial small subunit rDNA gene (mtSSU), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-alpha gene (TEF1). We also produced three more phylogenetic trees for Cantharellus based on 5.8S, nLSU, mtSSU, RPB2 and TEF1, Craterellus and Hydnum both based on the combined nLSU and ITS. This study has reproduced the status of Hydnaceae in the order Cantharellales, and phylogenetically confirmed seventeen genera in Hydnaceae. Twenty nine new taxa or synonyms are described, revealed, proposed, or reported, including eight new subgenera (Cantharellus subgenus Magnus, Craterellus subgenus Cariosi, subg. Craterellus, subg. Imperforati, subg. Lamelles, subg. Longibasidiosi, subg. Ovoidei, and Hydnum subgenus Brevispina); seventeen new species (Ca. laevihymeninus, Ca. magnus, Ca. subminor, Cr. badiogriseus, Cr. croceialbus, Cr. macrosporus, Cr. squamatus, H. brevispinum, H. flabellatum, H. flavidocanum, H. longibasidium, H. pallidocroceum, H. pallidomarginatum, H. sphaericum, H. tangerinum, H. tenuistipitum and H. ventricosum); two synonyms (Ca. anzutake and Ca. tuberculosporus as Ca. yunnanensis), and two newly recorded species (H. albomagnum and H. minum). The distinguishing characters of the new species and subgenera as well as their allied taxa are discussed in the notes which follow them. The delimitation and diversity in morphology, ecology, and phylogeny of Hydnaceae is discussed. Notes of seventeen genera which are phylogenetically accepted in Hydnaceae by this study and a key to the genera in Hydnaceae are provided.

Prenatal screening for trisomy 21: a comparative performance and cost analysis of different screening strategies.

BACKGROUND: Prenatal screening for chromosome aneuploidies have constantly been evolving, especially with the introduction of cell-free fetal DNA (cfDNA) screening in the most recent years. This study compares the performance, costs and timing of test results of three cfDNA screening implementation strategies: contingent, reflex and primary. METHODS: We modelled enhanced first trimester screening (eFTS) as the first-tier test in contingent or reflex strategies. cfDNA test was performed contingent on or reflex from eFTS results. A comparison was made between cfDNA screening using sequencing technology and Rolling Circle Amplification (RCA)/imaging solution. All model assumptions were based on results from previous publications or information from the Ontario prenatal screening population. RESULTS: At an eFTS risk cut-off of ≥1/1000, contingent and reflex cfDNA screening have the same detection rate (DR) (94%) for trisomy 21. Reflex cfDNA screening using RCA/Imaging solution provided the lowest false positive rate and cost. The number of women requiring genetic counselling and diagnostic testing was significantly reduced and women received their cfDNA screening result 9 days sooner compared with the contingent model. While primary cfDNA screening improved the trisomy 21 DR by 3-5%, it was more costly and more women required diagnostic testing. CONCLUSION: Reflex cfDNA screening is the most cost-effective prenatal screening strategy. It can improve the efficiency of prenatal aneuploidy screening by reducing the number of patient visits and providing more timely results.

[What are the real purpose and scope of screening for aneuploidy?] / Quels sont l'objectif réel et la portée du dépistage des aneuploïdies ?

In France, the recommended method for Down syndrome screening is the first trimester combined test, the risk assessment, based on maternal age, ultrasound measurement of fetal nuchal translucency and maternal serum markers (free ß-hCG and PAPP-A). The Down syndrome detection rate is 78.7% at a screen positive rate of 5%. However, the best screening test is the integrated test using a combination of first trimester combined test and second trimester quadruple test (serum α-fetoprotein, human chorionic gonadotropin, unconjugated E3, and dimeric inhibin-A) and being able to achieve a detection rate for Down syndrome of approximately 96% at a screen-positive rate of 5%. In recent years, the isolation of small fragments of "fetal" cell-free DNA in the maternal blood dramatically changed the screening strategy paradigm allowing a Down syndrome detection rate and false positive rate of 99.2 and 0.09%, respectively. However, aneuploidy screening based on cell-free DNA presents two major limitations which must be taken into account because they considerably limit its benefit: (i) not every woman will receive an interpretable result and that those who fail to receive a result are at increased risk for fetal aneuploidy: whether an inconclusive result is treated as screen positive or screen negative affects the overall detection rate (sensitivity) and false-positive rate (specificity) of the test; (ii) the limited number of targeted aneuploidies (trisomies 21, 18, 13 and common sex chromosome aneuploidies) in contrast to conventional noninvasive screening which is also able to detect rare aneuploidies, duplications, deletions, and other structural rearrangements. Of course, genetic counseling has to include a discussion about benefits and limitations of aneuploidy screening based on cell-free DNA. However, it should not be considered as a new screening test to substitute for conventional noninvasive screening. Moreover, if the ultimate goal is to deliver the most information about potential risk of various chromosomal abnormalities associated with adverse perinatal outcomes, then current cell-free DNA screening strategies may not be the best approach. These data highlight the limitations of cell-free DNA screening and the importance of a clear and fair information during pretest genetic counseling about benefits and limitations of any prenatal noninvasive screening (whether conventional or by cell-free DNA), but also about risks and benefits of invasive diagnostic procedures (in first- or second-line), especially since the cytogenetic analysis with chromosomal microarray analysis has improved the detection of genome microdeletions and microduplications (variants of the copy number) that can not be detected by standard cytogenetic analysis.

Genetic Counseling for Patients Considering Screening and Diagnosis for Chromosomal Abnormalities.

With the introduction of cell-free DNA screening for fetal aneuploidy and chromosomal microarray for prenatal diagnostic testing, options for pregnant women have become increasingly complex. Discussions regarding options for prenatal testing for aneuploidy should occur prior to any testing and should include pertinent risks and benefits of each alternative test. There is no single screening or diagnostic test option that is the right choice for all patients; patient decisions should be based on each individual woman's values and preferences after a discussion of all options.

Gold nanoparticle modified capacitive sensor platform for multiple marker detection.

The detection and quantification of cancer biomarkers in human blood is crucial to diagnose patients in the early stage of a disease. The recent advances in biosensor technology can improve detection by reducing the application time and cost without an invasive approach. In this study, a highly sensitive, novel nanoparticle-modified capacitive sensor was developed for the detection of cancer markers. The current work mainly focused on developing a surface modification protocol for achieving higher sensitivity using Au-NPs. An interdigitated electrode (IDE) transducer was modified using gold nanoparticles (Au-NPs) for signal enhancement, the platform was initially optimized with a small size IL-6 protein and the methodology was then applied for multiple marker detection with the aim of precise disease diagnostics. Carcinoembryonic antigen (CEA) and epidermal growth factor receptor (hEGFR) could be successfully detected in the concentration range of 20-1000 pg mL(-1) while cancer antigen 15-3 (CA15-3) was detected in the range of 10-200 U mL(-1). These results show an increase of sensitivity by five-fold with respect to those not modified, demonstrating a highly sensitive and specific capacitive immunoassay that has a great potential for the use of early diagnosis of cancer disease.

Comparison of three summary statistics for ranking genes in genome-wide association studies.

Problems associated with insufficient power have haunted the analysis of genome-wide association studies and are likely to be the main challenge for the analysis of next-generation sequencing data. Ranking genes according to their strength of association with the investigated phenotype is one solution. To obtain rankings for genes, researchers can draw from a wide range of statistics summarizing the relationships between variants mapped to a gene and the phenotype. Hence, it is of interest to explore the performance of these statistics in the context of rankings. To this end, we conducted a simulation study (limited to genes of equal sizes) of three different summary statistics examining the ability to rank genes in a meaningful order. The weighted sum of squared marginal score test (Pan, 2009), RareCover algorithm (Bahtia et al., 2010) and the elastic net regularization (Zou and Hastie, 2005) were chosen, because they can handle common as well as rare variants. The test based on the score statistic outperformed both other methods in almost all investigated scenarios. It was the only measure to consistently detect genes with interacting causal variants. However, the RareCover algorithm proved better at identifying genes including causal variants with small effect sizes and low minor allele frequency than the weighted sum of squared marginal score test. The performance of the elastic net regularization was unimpressive for all but the simplest scenarios.

Multiple Genetic Marker Analysis wih Using Quantitative RT-PCR in Gastric Cancer

PURPOSE: This study was aimed at evaluating the diagnostic validity of peritoneal dissemination of gastric cancer cells by performing multiple genetic marker analysis via quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in gastric cancer cell lines and gastric cancer tissues. MATERIALS AND METHODS: Quantitative RT-PCR was performed on 12 human gastric cancer cell lines and 10 gastric cancer tissues with four mRNAs of carcinoembryonic antigen (CEA), Cytokeratin 20 (CK20), dopa decarboxylase (DDC), and L-3-phosphoserine phosphatase (L3PP). RESULTS: Out of the 12 human gastric cancer cell lines we tested, CEA was overexpressed in four cell lines (33%), CK20 in one (8%), DDC in six (50%) and L3PP was expessed in all the lines (100%). Out of the 10 gastric cancer tissues we tested, CEA was overexpressed in nine tissues, CK20 in eight, DDC in nine and L3PP was overexpressed in all the tissues. L3PP was overexpressed in all the gastric cancer cell lines and tissues, but the levels of overexpression were lower than those of CEA and DDC. CONCLUSION: Multiple genetic marker analysis can compensate for the weak points of single marker analysis when testing gastric cancer, and three mRNAs of CEA, DDC and L3PP can be used as candidate genes.

Prenatal diagnosis for paediatricians.

In Ontario, approximately 140,000 women deliver newborn infants each year. Of these women, 60,000 to 70,000 have multiple marker screening, 10,000 undergo amniocentesis or chorion villus sampling and virtually all have at least one prenatal ultrasound. Multiple marker screening is not used in every province and territory; however, amniocentesis and prenatal ultrasound are used throughout Canada. Most paediatric patients will have been exposed to some form of prenatal diagnosis. If an abnormality is found prenatally, parents may have concerns to discuss with the paediatrician after the child is born. Likewise, if a child with a problem is born following a normal pregnancy, the parents will want to know why the problem was missed prenatally. Paediatricians should be aware of prenatal tests that have been performed to understand better their patients and their families.