Hatim T. Allawi

An invasive cleavage assay for direct quantitation of specific RNAs

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5′-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)–based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in ≥20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Invader Plus Method Detects Herpes Simplex Virus in Cerebrospinal Fluid and Simultaneously Differentiates Types 1 and 2

ABSTRACT We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.

Detection of Gastric Cancer with Novel Methylated DNA Markers: Discovery, Tissue Validation, and Pilot Testing in Plasma

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls. Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls. Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity. Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation‐specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele‐specific real‐time target and signal amplification assays on independent plasma‐extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long‐probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross‐validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six‐marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957, endothelin‐converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C‐type lectin domain containing 11A [CLEC11A]) normalized by beta‐1,3‐galactosyltransferase 6 (B3GALT6) level yielded a best‐fit AUC of 0.96 (95% CI, 0.93‐0.99) with HCC sensitivity of 95% (88%‐98%) at specificity of 92% (86%‐96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha‐fetoprotein (AFP) was 0.80 (0.74‐0.87) compared to 0.94 (0.9‐0.97) for the cross‐validated MDM panel (P < 0.0001). Conclusion: MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.

Abstract 712: Detection of lung cancer by assay of novel methylated DNA markers in plasma

Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2017-712

Erratum: An invasive cleavage assay for direct quantitation of specific RNAs

Peggy S. Eis, Marilyn C. Olson, Tsetska Takova, Michelle L. Curtis, Sarah M. Olson, Tatiana I. Vener, Hon S. Ip, Kevin L. Vedvik, Christian T. Bartholomay, Hatim T. Allawi, Wu-Po Ma, Jeff G. Hall, Michelle D. Morin, Tom H. Rushmore, Victor I. Lyamichev, and Robert W. Kwiatkowski. Nat. Biotechnol. 19, 673–676 (2001).