Methods for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer Detection: Application of Differential Centrifugation and Annexin A5 Coated Beads.

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads). The results showed that in a cultured medium, the majority of mRNA and lncRNA exist in larger EVs, whereas miRNA exist in both large and small EVs from the differential centrifugation fractions. All these RNA species exist in ANX beads captured EVs. We then used ANX beads to capture EVs in plasma samples from non-small-cell lung cancer patients and age-matched healthy volunteers. We found that the ANX bead capturing could efficiently improve RNA detection from human plasma, compared with direct extraction of RNA from plasma. Using ANX-bead capturing and reverse transcription and quantitative PCR, we detected significantly higher levels of CK19 mRNA, MALAT1 lncRNA, and miR155 miRNA in the plasma of lung cancer patients. These facts suggested the collection methods strongly affect the results of exRNA measurement from EVs, and that ANX beads can be a useful tool for detecting exRNA from plasma samples in clinical application.

Prenatal Diagnosis of Placental Mesenchymal Dysplasia with 46, X, Isochromosome Xq/45, X Mosaicism.

Placental mesenchymal dysplasia is an uncommon vascular anomaly of the placenta with characteristics of placentomegaly and multicystic appearance and with or without association with fetal chromosomal anomaly. We present a unique placental mesenchymal dysplasia patient with amniotic fluid karyotyping as 46, X, iso(X) (q10). Detailed molecular testing of the amniotic fluid, fetal cord blood, non-dysplastic placenta and dysplastic placenta was conducted after termination of pregnancy, from which we proved biparental/androgenetic (46, X, i(X) (q10)/45, X) mosaicism in different gestational tissues. A high portion of androgenetic cells in dysplastic placenta (74.2%) and near 100% of biparental cells in the fetus's blood and amniotic fluid were revealed. Delicate mosaic analyses were performed, and possible pathogenesis and embryogenesis of this case were drawn up.

Pathogenic Germline Mutations of DNA Repair Pathway Components in Early-Onset Sporadic Colorectal Polyp and Cancer Patients.

Given recent increases in the proportion of early-onset colorectal cancer (CRC), researchers are urgently working to establish a multi-gene screening test for both inherited and sporadic cancer-susceptible individuals. However, the incidence and spectrum of germline mutations in young sporadic CRC patients in East Asian countries and, especially, in sporadic polyp carriers and normal individuals are unknown. Peripheral blood samples were collected from 43 colonoscopy-proved normal controls and from 50 polyp patients and 49 CRC patients with no self-reported family history of cancer. All participants were under 50 years old. Next-generation sequencing with a panel of 30 CRC-associated susceptibility genes was employed to detect pathogenic germline mutations. The germline mutation carrier rates were 2.3%, 4.0%, and 12.2% in the normal, polyp, and cancer groups, respectively. A total of seven different mutations in six DNA repair pathway-related genes (MLH1, BRCA1, BRCA2, CHEK2, BLM, and NTHL1) were detected in nine participants. One frameshift mutation in BRCA2 and one frameshift mutation in the CHEK2 gene were found in a normal control and two colorectal polyp patients, respectively. One young sporadic CRC patient carried two heterozygous mutations, one in MLH1 and one in BRCA1. Three mutations (MLH1 p.Arg265Cys, MLH1 p.Tyr343Ter and CHEK2 p.Ile158TyrfsTer10) were each found in two independent patients and were considered "founder" mutations. This is the first report to demonstrate high percentage of germline mutations in young sporadic colorectal polyp, CRC, and general populations. A multi-gene screening test is warranted for the proactive identification of cancer-predisposed individuals.

Diagnosis of Pneumocystis pneumonia by real-time PCR in patients with various underlying diseases.

BACKGROUND: Pneumocystis pneumonia (PCP) is a disease caused by the opportunistic infection of the fungus Pneumocystis jirovecii. Several PCR methods have been developed to aid in the diagnosis of PCP. In this study, we evaluated the performance of a real-time PCR in the diagnosis of PCP, in patients with various underlying diseases. METHODS: Ninety-seven BAL samples and 94 sputum samples from 191 patients were used in the study. Patients were classified as PCP (121 patients) or non-PCP (70 patients) based on their clinical and radiological presentations. RESULTS: Real time PCR amplified the P. jirovecii mitochondrial large-subunit rRNA gene with a detection limit of 68 copies of DNA per reaction. Non-PCP pathogens including 32 different fungi and bacteria were also evaluated. Overall, 71.9% of the samples from PCP patients and 14.5% of those from non-PCP patients were positive for the PCR test with a CT value of the real-time PCR below 45. The main underlying diseases of the patients were hematological or solid malignancies (47.1%) and HIV infection (8.9%). The CT values of the test were significantly lower in BAL samples from PCP patients than those from non-PCP patients (p = 0.024). No non-PCP patient had a CT value below 30, whereas samples from 24.8% of PCP patients with underlying diseases had a CT value below 30. CONCLUSION: Since false positive PCR results were obtained, perhaps due to colonization, we suggest that the diagnosis of PCP should be based on a combination of clinical symptoms, underlying diseases, and PCR results.

A Simple and Highly Specific MassARRAY-Based Stool DNA Assay to Prioritize Follow-up Decisions in Fecal Immunochemical Test-Positive Individuals.

BACKGROUND: Seventy-five percent of fecal immunochemical test (FIT)-positive individuals are false positives and undergo unnecessary colonoscopies. Here, we established a stool DNA (sDNA) test that uses the Single Allele Base Extension Reaction (SABER) MassARRAY platform to improve the accuracy of FIT-based CRC detection. METHODS: Twenty-one variants in five CRC-associated genes were selected for the sDNA panel. Cell line DNA and matched mutation-confirmed tissue and stool samples from 34 patients were used for accuracy assessment (cohort 1). The clinical performance of the sDNA assay was further evaluated in 101 independent FIT-positive stool samples (cohort 2). RESULTS: In cohort 1, we obtained a 62% mutation concordance rate in paired tissue and stool samples of the CRC group, regardless of the FIT status. In cohort 2, 100% specificity in normal controls with positive FIT results was observed. By weighting the FIT value and the presence of a given variant type in stool and then summing the two scores, we found that a one-increment increase in the score was associated with a 4.538-fold risk (95% CI = 2.121⁻9.309) for malignancy in the FIT-positive setting. CONCLUSIONS: Our highly specific sDNA assay can help prioritize the most at-risk FIT-positive persons to receive prompt colonoscopic confirmation of CRC.

Acquired somatic TP53 or PIK3CA mutations are potential predictors of when polyps evolve into colorectal cancer.

Colorectal cancer (CRC) develops from accumulated mutations. However, which gene determines the malignant transformation from adenoma to carcinoma is still uncertain. Fifty-three formalin fixed paraffin-embedded polyps that had pathological findings from patients with hyperplasia, adenomatous, and tubular adenoma < 1 cm (non-neoplasia polyps, NNP, n = 27) or tubular adenoma ≥ 1 cm, tubulovillous and villous adenoma (neoplastic polyps, NP, n = 26) were recruited. Six paired synchronous polyps and cancer tissues and 50 independent fresh CRC tumors were also collected. All tissues were analyzed for their mutation genomes using next generation sequencing with a 50-gene panel. There were 40 types of somatic variants found in 7 genes, APC (43%), KRAS (28%), TP53 (11%), FBXW7 (8%), GNAS (4%), SMAD4 (2%), and BRAF (2%), and they were detected in 32 (60%) polyps. If combined with the mutation spectrum found in CRC tissues, a significant increase in the mutation rate in TP53 and PIK3CA from NNP, NP, early and late stage carcinoma (7%, 15%, 33.3% and 65% for TP53, p < 0.001; 0%, 0%, 23.3% and 25% for PIK3CA, p = 0.002) were noticed. Furthermore, distinct molecular features can be found in five pairs of synchronous polyps and tumors. However, TP53 or PIK3CA mutations can be found in tumor tissues but not in polyps. By systematically investigating the genome from polyps to tumor tissues, we demonstrated that acquired TP53 or PIK3CA somatic mutations are potential predictors for malignancy development. These results may aid in the identification of high risk individuals with tissues harboring mutations in these two genes.

Diagnosis of Tuberculosis Using Colorimetric Gold Nanoparticles on a Paper-Based Analytical Device.

We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10-2 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.

Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions.

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.

NRAS germline variant G138R and multiple rare somatic mutations on APC in colorectal cancer patients in Taiwan by next generation sequencing.

Colorectal cancer (CRC) arises from mutations in a subset of genes. We investigated the germline and somatic mutation spectrum of patients with CRC in Taiwan by using the AmpliSeq Cancer Hotspot Panel V2. Fifty paired freshly frozen stage 0-IV CRC tumors and adjacent normal tissue were collected. Blood DNA from 20 healthy donors were used for comparison of germline mutations. Variants were identified using an ion-torrent personal genomic machine and subsequently confirmed by Sanger sequencing or pyrosequencing. Five nonsynonymous germline variants on 4 cancer susceptible genes, CDH1, APC, MLH1, and NRAS, were observed in 6 patients with CRC (12%). Among them, oncogene NRAS G138R variant was identified as having a predicted damaging effect on protein function, which has never been reported by other laboratories. CDH1 T340A variants were presented in 3 patients. The germline variants in the cancer patients differed completely from those found in asymptomatic controls. Furthermore, a total of 56 COSMIC and 21 novel somatic variants distributed in 20 genes were detected in 44 (88%) of the CRC samples. High inter- and intra-tumor heterogeneity levels were observed. Nine rare variants located in the ß-catenin binding region of the APC gene were discovered, 7 of which could cause amino acid frameshift and might have a pathogenic effect. In conclusion, panel-based mutation detection by using a high-throughput sequencing platform can elucidate race-dependent cancer genomes. This approach facilitates identifying individuals at high risk and aiding the recognition of novel mutations as targets for drug development.

Real-time biallelic polymorphism-polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients.

BACKGROUND: An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP). METHODS: The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients. RESULTS: Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003-0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA. CONCLUSION: We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.

Use of X-linked short tandem repeats loci to confirm mutations in parentage caseworks.

BACKGROUND: Analysis of short tandem repeats (STRs) has become wide-spread in routine for parentage test. However, the accuracy of STR is sometimes interfered by the presence of microsatellite mutations. Analysis of other DNA markers such as the HV1 and HV2 hypervariable regions of mitochondrial DNA or the Y-STR becomes essential to settle the noncongruence. Owing to the time-consuming nature of these tests, we explored here the use of X-chromosome STR (X-STR) to resolve the paternity and maternity disputes. METHODS: At first, the autosomal STR mutation frequencies among 4758 Taiwanese were analyzed. Population data were obtained from randomly selected 99 females and 101 males to setup the X-STR database. Two families with a mismatch of one allele in autosomal STR analysis were subjected to the X-STR test to explore its clinical application. RESULTS: The STR mutations occurred in all 15 autosomal STR loci with the exception of TH01 and TPOX. The mutation rates could reach as high as 0.106% for the loci of D8S1179 and D18S51. As to the X-STR frequencies, the probability values of exact tests for Hardy-Weinberg equilibrium were 0.1471, 0.0019, 0.0025, 0.1427, and 0.1167 for the loci of DXS7132, DXS981, DXS6789, DXS101, and HPRTB, respectively. In addition, 33 and 34 different haplotypes were revealed for DXS101-DXS6789 and DXS7132-DXS981, respectively. Furthermore, two cases with one allele mismatch in routine parentage test were resolved by performing X-STR analysis. CONCLUSION: Typing of X-STR markers is recommended for parentage test when 1 or 2 alleles mismatch is present or when the samples are difficult to be analyzed.

A novel cis-AB allele derived from the A transferase gene by nucleotide substitution C796A.

The cis-AB is a very rare phenotype in the ABO blood group system. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B antigens. Until now, gene sequences of only 3 cis-AB alleles were characterized. One was the A(1v) allele with a nucleotide substitution G803C at codon 268; the second was the B allele with a nucleotide substitution A796C at codon 266; and the third arose from a point mutation C700T at codon 234 in exon 7 of the B transferase gene. In this study, we found a novel cis-AB allele when performing paternity tests in Chang Gung Memorial Hospital in Taiwan. Although his father was O blood type, a serologically AB blood type child was confirmed as being his father's offspring on the basis of 16 microsatellite markers (99.97% plausibility for the child and father). Exons 6 and 7 of the child's ABO alleles were characterized by direct sequencing and gene cloning. The results showed that the child has one O(1) allele and the second allele is almost identical to A(1*02) allele except for a single point mutation at nucleotide position 796, where an A replaces a C and leads to a change of leucine to methionine at amino acid 266. This implies that the child's O(1) allele was inherited from his father and the other allele was inherited from his mother. In conclusion, the novel cis-AB allele reported here is derived from the A transferase gene through a nucleotide substitution C796A, which differs from the 3 previously reported cis-AB alleles.