Prognostic Implications of Multiplex Detection of <i>KRAS</i> Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma

Kim, Min Kyeong,Woo, Sang Myung,Park, Boram,Yoon, Kyong-Ah,Kim, Yun-Hee,Joo, Jungnam,Lee, Woo Jin,Han, Sung-Sik,Park, Sang-Jae,Kong, Sun-Young American Association for Clinical Chemistry, Inc. 2018 Clinical chemistry Vol.64 No.4

<P><B>BACKGROUND:</B></P><P>Cell-free DNA (cfDNA) is known to provide potential biomarkers for predicting clinical outcome, but its value in pancreatic ductal adenocarcinoma (PDAC) has not been fully evaluated. The aim of this study was to evaluate the clinical applicability of quantitative analysis of multiplex <I>KRAS</I> mutations in cell-free DNA from patients with PDAC.</P><P><B>METHODS:</B></P><P>A total of 106 patients with PDAC were enrolled in this prospective study. The concentration and fraction of <I>KRAS</I> mutations were determined through multiplex detection of <I>KRAS</I> mutations in plasma samples by use of a droplet digital PCR kit (Bio-Rad).</P><P><B>RESULTS:</B></P><P><I>KRAS</I> mutations were detected in 96.1% of tissue samples. Eighty patients (80.5%) harbored <I>KRAS</I> mutations in cfDNA, with a median <I>KRAS</I> mutation concentration of 0.165 copies/μL and a median fractional abundance of 0.415%. Multivariable analyses demonstrated that the <I>KRAS</I> mutation concentration [hazard ratio (HR), 2.08; 95% CI, 1.20–3.63] and <I>KRAS</I> fraction (HR, 1.73; 95% CI, 1.02–2.95) were significant factors for progression-free survival. <I>KRAS</I> mutation concentration (HR, 1.97; 95% CI, 1.05–3.67) also had prognostic implications for overall survival. Subgroup analyses showed that <I>KRAS</I> mutation concentration and fractional abundance significantly affected progression-free survival in resectable PDAC (<I>P</I> = 0.016). Moreover, when combined with the cancer biomarker CA19-9, the <I>KRAS</I> mutation concentration in cfDNA showed additive benefits for the prediction of overall survival.</P><P><B>CONCLUSIONS:</B></P><P>This study demonstrates that multiplex detection of <I>KRAS</I> mutations in plasma cfDNA is clinically relevant, providing a potential candidate biomarker for prognosis of PDAC.</P>

Rapid Diagnosis of Tick-Borne Illnesses by Use of One-Step Isothermal Nucleic Acid Amplification and Bio-Optical Sensor Detection

Kim, Ji Yeun,Koo, Bonhan,Jin, Choong Eun,Kim, Min Chul,Chong, Yong Pil,Lee, Sang-Oh,Choi, Sang-Ho,Kim, Yang Soo,Woo, Jun Hee,Shin, Yong,Kim, Sung-Han American Association for Clinical Chemistry, Inc. 2018 Clinical chemistry Vol.64 No.3

<P><B>BACKGROUND:</B></P><P>Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the most common tick-borne illnesses in South Korea. Early differentiation of SFTS from scrub typhus in emergency departments is essential but difficult because of their overlapping epidemiology, shared risk factors, and similar clinical manifestations.</P><P><B>METHODS:</B></P><P>We compared the diagnostic performance of one-step isothermal nucleic acid amplification with bio-optical sensor detection (iNAD) under isothermal conditions, which is rapid (20–30 min), with that of real-time PCR, in patients with a confirmed tick-borne illness. Fifteen patients with confirmed SFTS who provided a total of 15 initial blood samples and 5 follow-up blood samples, and 21 patients with confirmed scrub typhus, were evaluated.</P><P><B>RESULTS:</B></P><P>The clinical sensitivity of iNAD (100%; 95% CI, 83–100) for SFTS was significantly higher than that of real-time PCR (75%; 95% CI, 51–91; <I>P</I> = 0.047), while its clinical specificity (86%; 95% CI, 65–97) was similar to that of real-time PCR (95%; 95% CI, 77–99; <I>P</I> = 0.61). The clinical sensitivity of iNAD for scrub typhus (100%; 95% CI, 81–100) was significantly higher than that of real-time PCR for scrub typhus (67%; 95% CI, 43–85; <I>P</I> = 0.009), while its clinical specificity (90%; 95% CI, 67–98) was similar to that of real-time PCR (95%; 95% CI, 73–100; <I>P</I> > 0.99).</P><P><B>CONCLUSIONS:</B></P><P>iNAD is a valuable, rapid method of detecting SFTS virus and <I>Orientia tsutsugamushi</I> with high clinical sensitivity and specificity.</P>

Clinical Assay for AFP-L3 by Using Multiple Reaction Monitoring–Mass Spectrometry for Diagnosing Hepatocellular Carcinoma

Kim, Hyunsoo,Sohn, Areum,Yeo, Injoon,Yu, Su Jong,Yoon, Jung-Hwan,Kim, Youngsoo American Association for Clinical Chemistry, Inc. 2018 Clinical chemistry Vol.64 No.8

<P><B>BACKGROUND:</B></P><P><I>Lens culinaris</I> agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring–mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum.</P><P><B>METHODS:</B></P><P>The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using <I>L. culinaris</I> agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation.</P><P><B>RESULTS:</B></P><P>The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines.</P><P><B>CONCLUSIONS:</B></P><P>Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.</P>

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer

Li, Feng,Yoshizawa, Janice M.,Kim, Kyoung-Mee,Kanjanapangka, Julie,Grogan, Tristan R.,Wang, Xiaoyan,Elashoff, David E.,Ishikawa, Shigeo,Chia, David,Liao, Wei,Akin, David,Yan, Xinmin,Lee, Min-Sun,Choi, American Association for Clinical Chemistry, Inc. 2018 Clinical chemistry Vol.64 No.10

<P><B>BACKGROUND:</B></P><P>Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC.</P><P><B>METHODS:</B></P><P>Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel.</P><P><B>RESULTS:</B></P><P>We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (<I>SPINK7</I>, <I>PPL</I>, and <I>SEMA4B</I>) and 2 miRNAs (<I>MIR140-5p</I> and <I>MIR301a</I>), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72–0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80–0.93).</P><P><B>CONCLUSIONS:</B></P><P>We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.</P>

Rapid One-Step Plasma Test for the Electrochemical and Colorimetric Detection of a Universal Cancer Biomarker

American Association for Clinical Chemistry, Inc. 2019 Clinical chemistry Vol.65 No.7

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

Whale, Alexandra S.,Jones, Gerwyn M.,Pavš,ič,, Jernej,Dreo, Tanja,Redshaw, Nicholas,Akyü,rek, Sema,Akgö,z, Mü,slü,m,Divieto, Carla,Sassi, Maria Paola,He, Hua-Jun,Cole, Kennet American Association for Clinical Chemistry, Inc. 2018 Clinical chemistry Vol.64 No.9

<P><B>BACKGROUND:</B></P><P>Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.</P><P><B>METHODS:</B></P><P>We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (<I>KRAS</I> c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement.</P><P><B>RESULTS:</B></P><P>Concentration values for samples of <I>KRAS</I> G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%–8% and 5%–10%, respectively).</P><P><B>CONCLUSIONS:</B></P><P>This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.</P>

Noninvasive Prenatal Diagnosis of Duchenne Muscular Dystrophy: Comprehensive Genetic Diagnosis in Carrier, Proband, and Fetus

Yoo, Seong-Keun,Lim, Byung Chan,Byeun, Jiyoung,Hwang, Hee,Kim, Ki Joong,Hwang, Yong Seung,Lee, JoonHo,Park, Joong Shin,Lee, Yong-Sun,Namkung, Junghyun,Park, Jungsun,Lee, Seungbok,Shin, Jong-Yeon,Seo, American Association for Clinical Chemistry, Inc. 2015 Clinical chemistry Vol.61 No.6

<P><B>BACKGROUND:</B></P><P>Noninvasive prenatal diagnosis of monogenic disorders using maternal plasma and targeted massively parallel sequencing is being investigated actively. We previously demonstrated that comprehensive genetic diagnosis of a Duchenne muscular dystrophy (DMD) patient is feasible using a single targeted sequencing platform. Here we demonstrate the applicability of this approach to carrier detection and noninvasive prenatal diagnosis.</P><P><B>METHODS:</B></P><P>Custom solution-based target enrichment was designed to cover the entire dystrophin (<I>DMD</I>) gene region. Targeted massively parallel sequencing was performed using genomic DNA from 4 mother and proband pairs to test whether carrier status could be detected reliably. Maternal plasma DNA at varying gestational weeks was collected from the same families and sequenced using the same targeted platform to predict the inheritance of the <I>DMD</I> mutation by their fetus. Overrepresentation of an inherited allele was determined by comparing the allele fraction of 2 phased haplotypes after examining and correcting for the recombination event.</P><P><B>RESULTS:</B></P><P>The carrier status of deletion/duplication and point mutations was detected reliably through using a single targeted massively parallel sequencing platform. Whether the fetus had inherited the <I>DMD</I> mutation was predicted correctly in all 4 families as early as 6 weeks and 5 days of gestation. In one of these, detection of the recombination event and reconstruction of the phased haplotype produced a correct diagnosis.</P><P><B>CONCLUSIONS:</B></P><P>Noninvasive prenatal diagnosis of DMD is feasible using a single targeted massively parallel sequencing platform with tiling design.</P>