Improving Fetal Fraction of Noninvasive Prenatal Screening Samples Collected in EDTA-Gel Tubes Using Gel Size Selection: A Head-To-Head Comparison of Methods.

The aim of this study was to compare the use of EDTA-gel blood collection tubes with and without size selection to cell-stabilizing collection tubes for remote blood sampling for noninvasive prenatal screening (NIPS). Sixty-one pregnant women at 10 to 14 weeks' gestation undergoing NIPS were recruited. Participants were phlebotomized with Streck and EDTA-gel tubes. EDTA-gel tubes were centrifuged before shipping. Libraries prepared from cell-free DNA (cfDNA) extracted from both types of tubes were sequenced on Illumina NextSeq 500, and fetal fraction was estimated using SeqFF. EDTA-gel tube libraries were size selected on agarose gel to eliminate cfDNA fragments >160 bp and resequenced. The main outcome measure was fetal fraction expressed as percentage of total cfDNA sequenced, calculated from sequence read counts (SeqFF). Streck tube samples showed an average 1% higher fetal fraction than centrifuged EDTA-gel tubes without size selection. This difference increased with temperature. When EDTA-gel samples' libraries were size selected, the mean fetal fraction increased from 7% to 13%, with no sample having fetal fraction <4%. Using EDTA-gel tubes reduces NIPS sampling cost and tube processing time in the laboratory. Also, using EDTA-gel tubes does not lead to cfDNA degradation. Size selection increases fetal fraction, reduces the number of test failures, increases NIPS clinical performance, and may be helpful in situations asking for a higher fetal fraction, such as twin pregnancies or screening for sub-chromosomal imbalances.

Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening.

OBJECTIVES: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. METHODS: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. RESULTS: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. CONCLUSIONS: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Validation of a New Protocol to Collect and Isolate Plasma from Pregnant Women for Noninvasive Prenatal Testing (NIPT).

BACKGROUND: Most laboratories use specialized tubes (e.g., Streck) to recover circulating cell-free DNA (ccfDNA) for noninvasive prenatal testing (NIPT). We validated a low cost, simple procedure for collecting NIPT samples in remote laboratories that avoids highspeed centrifugation. EDTA gel blood sampling tube allows simple separation of plasma from blood cells. Decanted plasma is filtered to remove cell debris. The procedure can be performed within a few minutes after the blood centrifugation step, and ccfDNA-grade plasma can be frozen for transportation. METHODS: We recruited 51 pregnant women and collected blood in one EDTA-gel Greiner tube and two Streck tubes. All tubes were centrifuged at 1600 g x 10 min within 6 h of sample collection. Plasma from EDTA tubes was poured into a syringe cylinder and filtered through a 0.45 µm Millipore filter. Plasma from Streck tubes was recovered with a pipette and one was filtered as above while the second was centrifuged at 16 000 g. The ccfDNA was isolated and NGS sequencing libraries were prepared and sequenced on an Illumina system. Fetal fractions were estimated using SeqFF. This study had a power of 79% to detect a decrease of 1% in fetal fractions with the new method. RESULTS: We did not observe any significant difference between the three procedures for the fetal fraction nor for the quality or quantity of libraries produced. CONCLUSION: EDTA-gel tubes with filtration provide high quality plasma for ccfDNA analysis and can be sent frozen to the NIPT laboratory. This is economical and it frees the laboratory of time-consuming steps.

Precision of Fetal DNA Fraction Estimation by Quantitative Polymerase Chain Reaction Quantification of a Differently Methylated Target in Noninvasive Prenatal Testing.

BACKGROUND: The performance of noninvasive prenatal testing (NIPT) assays is critically determined by the proportion of fetal DNA or fetal fraction (FF). Fetomaternal differential methylation of certain genomic regions has been proposed as a universal marker of fetal origin, and previous reports have suggested the use of methylation-sensitive restriction enzyme (MSRE) assays to estimate FF. METHODS: We analyzed the performance of FF estimation using an MSRE assay with duplex quantitative polymerase chain reaction (qPCR). Mixtures of genomic DNA from placental cells and from adult women were digested with 2 MSRE and FF estimates obtained, for a total of 221 pairwise treatment/control comparisons. RESULTS: The coefficient of variance (CV) of the MSRE assays was high, ranging from 24% to 60%. An alternative in silico FF estimation algorithm, SeqFF, displayed slightly lower variability, with a CV of 22%. CONCLUSION: These results cast doubts on the usefulness of the MSRE-based assay of differentially methylated markers for FF estimation. The lack of a universal method capable of precisely estimating FF remains an incompletely solved issue.

Development of Reference Materials for Noninvasive Prenatal Aneuploidy Testing by Massively Parallel Sequencing: A Proof-of-Concept Study.

BACKGROUND: Noninvasive prenatal aneuploidy testing (NIPT) represents the first large-scale clinical application of massively parallel sequencing technology. However, no NIPT reference material (RM) has yet been widely adopted, impeding the development of quality management systems and standardization. Developing an NIPT RM from a biological sample is complicated by the low concentration of cell-free DNA (cfDNA), which implies pooling specimens and frequent resampling. METHODS: We tested the feasibility of using DNA from immortalized cell lines of a woman and her aneuploid offspring to spike an artificial plasma matrix. Enzymatic fragmentation of extracted DNA was optimized to achieve fragment size profiles with a mode of 150 to 200 bp, similar to biological cfDNA. This synthetic material was compared with routine biological samples from pregnant women by a targeted NIPT assay in a multiplex sequencing run on a Proton platform. RESULTS: Sequencing statistics were similar between artificially prepared material and routine biological samples, as well as relative chromosomal representation, and no matrix effects could be detected. Estimate of fetal fraction (FF) was within the range of expected value, and aneuploidy detection statistic (z-score) was also comparable between both types of samples. CONCLUSIONS: Artificial plasma spiked with DNA from cell lines of mother and offspring is a promising strategy for developing NIPT RM. This type of material would offer the advantage of a constant and stable composition, allowing for greater standardization of NIPT assays. Moreover, it preserves the parental relatedness used by targeted assay to estimate FF by identification of paternal alleles in single-nucleotide polymorphisms or other variable regions.

Prospective head-to-head comparison of accuracy of two sequencing platforms for screening for fetal aneuploidy by cell-free DNA: the PEGASUS study.

We compared clinical validity of two non-invasive prenatal screening (NIPS) methods for fetal trisomies 13, 18, 21, and monosomy X. We recruited prospectively 2203 women at high risk of fetal aneuploidy and 1807 at baseline risk. Three-hundred and twenty-nine euploid samples were randomly removed. The remaining 1933 high risk and 1660 baseline-risk plasma aliquots were assigned randomly between four laboratories and tested with two index NIPS tests, blind to maternal variables and pregnancy outcomes. The two index tests used massively parallel shotgun sequencing (semiconductor-based and optical-based). The reference standard for all fetuses was invasive cytogenetic analysis or clinical examination at birth and postnatal follow-up. For each chromosome of interest, chromosomal ratios were calculated (number of reads for chromosome/total number of reads). Euploid samples' mean chromosomal ratio coefficients of variation were 0.48 (T21), 0.34 (T18), and 0.31 (T13). According to the reference standard, there were 155 cases of T21, 49 T18, 8 T13 and 22 45,X. Using a fetal fraction ≥4% to call results and a chromosomal ratio z-score of ≥3 to report a positive result, detection rates (DR), and false positive rates (FPR) were not statistically different between platforms: mean DR 99% (T21), 100%(T18, T13); 79%(45,X); FPR < 0.3% for T21, T18, T13, and <0.6% for 45,X. Both methods' negative predictive values in high-risk pregnancies were >99.8%, except for 45,X(>99.6%). Threshold analysis in high-risk pregnancies with different fetal fractions and z-score cut-offs suggested that a z-score cutoff to 3.5 for positive results improved test accuracy. Both sequencing platforms showed equivalent and excellent clinical validity.

Non-invasive prenatal aneuploidy testing: Critical diagnostic performance parameters predict sample z-score values.

OBJECTIVES: Non-invasive prenatal aneuploidy testing (NIPT) by next-generation sequencing of circulating cell-free DNA in maternal plasma relies on chromosomal ratio (chrratio) measurements to detect aneuploid values that depart from euploid ratios. Diagnostic performances are known to depend on the fraction of fetal DNA (FF) present in maternal plasma, although how this translates into specific quantitative changes in specificity/positive predictive values and which other variables might also be important is not well understood. DESIGN & METHODS: To explore this issue, theoretical relationships between FF and various measures of diagnostic performances were assessed for a range of parameter values. Empirical data from three NIPT assays were then used to validate theoretical calculations. RESULTS: For a given positivity threshold, dramatic changes in specificity and positive predictive values (PPV) as a function of both FF and the coefficient of variation (CV) of the chrratio measurement were observed. Theoretically predicted and observed chrratio z-scores agreed closely, confirming the determinant impact of small changes in both FF and chrratio CV. CONCLUSIONS: Evaluation of NIPT assay performances therefore requires knowledge of the FF distribution in the population in which the test is intended to be used and, in particular, of the precise value of the assay chrratio CV for each chromosome or genomic region of interest. Laboratories offering NIPT testing should carefully measure these parameters to ensure test reliability and clinical usefulness in interpreting individual patients' results.

Prognostic value of urinary prostate cancer antigen 3 (PCA3) during active surveillance of patients with low-risk prostate cancer receiving 5α-reductase inhibitors.

OBJECTIVES: To determine the clinical performance of the urinary prostate cancer antigen 3 (PCA3) test to predict the risk of Gleason grade re-classification amongst men receiving a 5α-reductase inhibitor (5ARI) during active surveillance (AS) for prostate cancer. PATIENTS AND METHODS: Patients with low-risk prostate cancer were enrolled in a prospective Phase II study of AS complemented with prescription of a 5ARI. A repeat biopsy was taken within the first year and annually according to physician and patient preference. In all, 90 patients had urine collected after digital rectal examination of the prostate before the first repeat biopsy. The PCA3 test was performed in a blinded manner at a central laboratory. RESULTS: Using a PCA3-test score threshold of 35, there was a significant difference (P < 0.001) in the risk of being diagnosed with Gleason ≥7 cancer during a median of 7 years of follow-up. Adjusted Cox regression and Kaplan-Meier analyses also showed a significantly higher risk of upgrading to Gleason ≥7 during follow-up for those with a higher PCA3-test score. CONCLUSION: The urinary PCA3 test predicted Gleason grade re-classification amongst patients receiving a 5ARI during AS for low-risk prostate cancer.

Comparison of first-tier cell-free DNA screening for common aneuploidies with conventional publically funded screening.

OBJECTIVE: This study evaluates the impact of offering cell-free DNA (cfDNA) screening as a first-tier test for trisomies 21 and 18. METHODS: This is a prospective study of pregnant women undergoing conventional prenatal screening who were offered cfDNA screening in the first trimester with clinical outcomes obtained on all pregnancies. RESULTS: A total of 1198 pregnant women were recruited. The detection rate of trisomy 21 with standard screening was 83% with a false positive rate (FPR) of 5.5% compared with 100% detection and 0% FPR for cfDNA screening. The FPR of cfDNA screening for trisomies 18 and 13 was 0.09% for each. Two percent of women underwent an invasive diagnostic procedure based on screening or ultrasound findings; without the cfDNA screening, it could have been as high as 6.8%. Amongst the 640 women with negative cfDNA results and a nuchal translucency (NT) ultrasound, only 3 had an NT greater or equal to 3.5 mm: one had a normal outcome and two lost their pregnancy before 20 weeks. CONCLUSIONS: cfDNA screening has the potential to be a highly effective first-tier screening approach leading to a significant reduction of invasive diagnostic procedures. For women with a negative cfDNA screening result, NT measurement has limited clinical utility.

Phase II Drug-Metabolizing Polymorphisms and Smoking Predict Recurrence of Non-Muscle-Invasive Bladder Cancer: A Gene-Smoking Interaction.

Cigarette smoking is the most important known risk factor for urinary bladder cancer. Selected arylamines in cigarette smoke are recognized human bladder carcinogens and undergo biotransformation through several detoxification pathways, such as the glutathione S-transferases (GST), and uridine-diphospho-glucuronosyltransferases (UGT) pathways. GSTM1 deletion status and UGT1A1*28 rs8175347 genotypes were assessed in 189 non-muscle-invasive bladder cancers (NMIBC) patients with pTa (77.2%) and pT1 (22.8%) tumors and a mean follow-up of 5.6 years, to investigate whether two common functional polymorphisms in GSTM1 and UGT1A1 genes and smoking history are associated with recurrence-free survival of patients with NMIBC. Most patients were current (48.7%) or previous (35.4%) cigarette smokers and 15.9% never smoked. Tumor recurrence occurred in 65.1% of patients, at a median time of 12.9 months. Upon multivariate analysis, previous and current smokers approximately tripled their risk of recurrences [HR = 2.76; 95% confidence interval (CI), 1.03-7.40 and HR = 2.93; 95% CI, 1.08-7.94, respectively]. When adjusted for age, smoking status, stage, grade, gender, and presence of carcinoma in situ, carriers of GSTM1 (+/- and -/-) and UGT1A1*28/*28 alleles were significantly at risk of NMIBC recurrence (HR = 10.05; 95% CI, 1.35-75.1 and HR = 1.91; 95% CI, 1.01-3.62, respectively). Compared with nonsmokers with UGT1A1*1/*1 and *1/*28 genotypes, previous and current smokers homozygous for the UGT1A1*28 allele demonstrated a risk of recurrence of 4.95 (95% CI, 1.02-24.0) and 5.32 (95% CI, 2.07-13.7), respectively. This study establishes a connection between GSTM1, UGT1A1, and tobacco exposure as prognostic markers of NMIBC recurrence in bladder cancer patients. These findings warrant validation in larger cohorts.

Comparison of digital image analysis and visual scoring of KI-67 in prostate cancer prognosis after prostatectomy.

BACKGROUND: The tumor proliferative index marker Ki-67 was shown to be associated with clinically significant outcomes in prostate cancer, but its clinical application has limitations due to lack of uniformity and consistency in quantification. Our objective was to compare the measurements obtained with digital image analysis (DIA) versus virtual microscopy (visual scoring (VS)). METHODS: To do so, we compared the measurement distributions of each technique and their ability to predict clinically useful endpoints. A tissue microarray series from a cohort of 225 men who underwent radical prostatectomy was immunostained for Ki-67. The percentage of Ki-67 positive nuclei in malignant cells was assessed both by VS and DIA, and a H-score was calculated. The distribution and predictive ability of these scoring methods to predict biochemical recurrence (BCR) and death from prostate cancer (DPCa) were compared using Mann-Whitney test and C-index. RESULTS: The measurements obtained with VS were similar to the DIA measurements (p = 0.73) but dissimilar to the H-score (p < 0.001). Cox regression models showed that Ki-67 was associated with BCR (HR 1.46, 95 % CI 1.10-1.94) and DPCa (HR 1.26, 95 % CI 1.06-1.50). C-indexes revealed that Ki-67 was a better predictor of DPCa (0.803, 0.8059 and 0.789; VS, DIA and H-score, respectively) than of BCR (0.625, 0.632 and 0.604; VS, DIA and H-score, respectively). CONCLUSION: The measurement distributions and the predictive abilities of VS and DIA were similar and presented the same predictive behaviour in our cohort, supporting the role of Ki-67 proliferative index as an important prognostic factor of BCR and DPCa in prostate cancer post RP. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6656878501536663.

FDG-PET/CT for pre-operative staging and prognostic stratification of patients with high-grade prostate cancer at biopsy.

BACKGROUND: The role of (18)F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in prostate cancer (PCa) has not been well defined yet. Because high-grade PCa tends to exhibit increased glycolytic rate, FDG-PET/CT could be useful in this setting. The aim of this study was to assess the value of FDG-PET/CT for pre-operative staging and prognostic stratification of patients with high-grade PCa at biopsy. METHODS: Fifty-four patients with a Gleason sum≥8 PCa at biopsy underwent FDG-PET/CT as part of the staging workup. Thirty-nine patients underwent radical prostatectomy (RP) and pelvic lymph node (LN) dissection, 2 underwent LN dissection only, and 13 underwent non-surgical treatments. FDG-PET/CT findings from clinical reports, blinded reading and quantitative analysis were correlated with clinico-pathological characteristics at RP. RESULTS: Suspicious foci of increased FDG uptake were found in the prostate, LNs and bones in 44, 13 and 6% of patients, respectively. Higher clinical stage, post-RP Gleason sum and pattern, and percentage of cancer involvement within the prostate were significantly associated with the presence of intraprostatic FDG uptake (IPFU) (P<0.05 in all cases). Patients without IPFU who underwent RP were downgraded to Gleason≤7 in 84.6% of cases, as compared to 30.8% when IPFU was reported (P=0.003). Qualitative and quantitative IPFU were significantly positively correlated with post-RP Gleason pattern and sum, and pathological T stage. Absence and presence of IPFU were associated with a median 5-year cancer-free survival probability of 70.2 and 26.9% (P=0.0097), respectively, using the CAPRA-S prognostic tool. CONCLUSION: These results suggest that, among patients with a high-grade PCa at biopsy, FDG-PET/CT could improve pre-treatment prognostic stratification by predicting primary PCa pathological grade and survival probability following RP.

Prostatic and dietary omega-3 fatty acids and prostate cancer progression during active surveillance.

The association between omega-3 (ω-3) fatty acids and prostate cancer has been widely studied. However, little is known about the impact of prostate tissue fatty acid content on prostate cancer progression. We hypothesized that compared with the estimated dietary ω-3 fatty acids intake and the ω-3 fatty acids levels measured in red blood cells (RBC), the prostate tissue ω-3 fatty acid content is more strongly related to prostate cancer progression. We present the initial observations from baseline data of a phase II clinical trial conducted in a cohort of 48 untreated men affected with low-risk prostate cancer, managed under active surveillance. These men underwent a first repeat biopsy session within 6 months after the initial diagnosis of low-risk prostate cancer, at which time 29% of the men had progressed from a Gleason score of 6 to a Gleason score of 7. At the first repeat biopsy session, fatty acid levels were assessed with a food-frequency questionnaire, and determined in the RBC and in the prostate tissue biopsy. We found that eicosapentaenoic acid (EPA) was associated with a reduced risk of prostate cancer progression when measured directly in the prostate tissue. Thus, this initial interim study analysis suggests that prostate tissue ω-3 fatty acids, especially EPA, may be protective against prostate cancer progression in men with low-risk prostate cancer.

High level of mature tumor-infiltrating dendritic cells predicts progression to muscle invasion in bladder cancer.

A prognostic value for tumor-associated macrophages (TAMs) and tumor-infiltrating dendritic cells (TIDCs) has been reported in many human cancers. The objective of this study is to determine the prognostic value of CD83(+) mature TIDCs and CD68(+) TAMs in non-muscle-invasive bladder cancer at first diagnosis. Immunohistochemistry staining was performed with anti-CD68 and anti-CD83 monoclonal antibodies on tissue sections from 93 formalin-fixed, paraffin-embedded tissue blocks from pTa and pT1 bladder tumors. A scoring index based on the average density of observed positive cells in the papillary axis, the stroma, lymphoid aggregates, and into tumor foci was calculated for each patient. Comparison of baseline characteristics with marker levels was done using Pearson χ(2) or Fisher exact test. Kaplan-Meier analyses and Cox regression models were fitted to evaluate the prognostic value of TIDCs and TAMs. The absence of both CD68(+) TAMs and CD83(+) TIDCs was associated with tumor recurrence. The presence of TIDCs was associated with a significant risk of progression to muscle-invasive cancer (hazard ratio, 8.253; P = .0179). Patients were risk stratified using age and TIDC score. Patients 70 years or older with a high score of TIDCs had a 56% progression-free survival after 6 years compared with 94% for patients younger than 70 years with a low score of TIDCs. None of 20 patients with a low score of TAMs progressed. These data indicate that the presence of mature TIDCs and possibly TAMs may help risk-stratify patients at the time of first diagnosis of non-muscle-invasive bladder cancer and may be useful in tailoring follow-up and treatment strategies.

Image multidistortion estimation.

We present a method for estimating the amount of noise and blur in a distorted image. Our method is based on the multiscale structural similarity (MS-SSIM) framework that, although designed to measure image quality, is used to estimate the amount of blur and noise in a degraded image given a reference image. We show that there exists a bijective mapping between the 2-D noise/blur space and the 3-D MS-SSIM space, which allows to recover distortion parameters. That mapping allows to formulate the multidistortion-estimation problem as a classical optimization problem. Various search strategies such as Newton, simplex, NewUOA, and brute-force search are presented and compared. We also show that a bicubic patch can be used to approximate the bijective mapping between the noise/blur space and the 3-D MS-SSIM space. Interestingly, the use of such a patch reduces the processing time by a factor of 40 without significantly reducing precision. Based on quantitative results, we show that the amount of different types of blur and noise in a distorted image can be recovered with accuracy of roughly 2% and 8%, respectively. Our methods are compared with four state-of-the-art noise- and blur-estimation techniques.

Allergy to multiple local anesthetics.

Real allergy to local anesthetic (LA) is very rare. This study was performed to report a case of anaphylaxis to multiple "caine." A 25-years-old atopic nurse developed a very severe anaphylactic reaction on her third infiltration for low back pain with bupivacaine, lidocaine, and methylprednisolone: she developed a vagal reaction, followed during the next 30 minutes by a pruriginous skin rash, followed by a tongue edema and a severe bronchospasm. Adrenalin was injected with a poor response. She was intubated and transferred to the intensive care unit for a few days and, finally, she recuperated completely. Skin-prick tests were done on two occasions. In the first session, no reactions were observed with triamcinolone and methylprednisolone at 1 mg/cc, but a rapid extending maculopapular erythema developed with a final diameter of 50 mm with lidocaine 0.1% (group 2) and 25 mm with procaine 2% (group 1): control 0 mm, histamine, 3 mm. She also complained of itchiness in the neck and shoulder, which resolved in the next 90 minutes. In the second session, a test with bupivacaine 0.0005% (group 2) gave a papule with a diameter of >5 mm, and a test with mepivacaine 0.001% (group 2) was negative: control, histamine, 3 mm; no subsequent tests with mepivacaine were done because she developed a cough and throat pruritus, voice modification, and a sensation of throat narrowing, that resolved with treatment. We reported a case of anaphylaxis to multiple LA (groups 1 and 2), possibly via an IgE-mediated mechanism.