Lauren M. Stanoszek

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

A New StaRT-PCR Approach to Detect and Quantify Fish Viral Hemorrhagic Septicemia Virus (VHSv): Enhanced Quality Control with Internal Standards

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the worlds most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

Background Reverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed. Methods We developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples. Results Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples. Conclusions This quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

Reliable breakpoint cluster region (BCR)--Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 10(3) GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 10(3) GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.

Abstract 61: Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer.

Quantitative RT-PCR tests that measure transcript abundance of selected genes in clinical specimens promise to improve cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer. However, most clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE) and these yield sub-optimal RNA integrity. Thus, there is a need for qPCR tests with increased robustness that also have intrinsic quality control and low cost. To address this need, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of gene-specific internal standard (IS) molecules using two-color fluorometric analysis on real-time platform. For each gene, a competitive template IS was synthesized containing 4-6 nucleotide changes relative to the native template (NT). During PCR, NT or IS signal was quantified with sequence-specific FAM-labeled probe or Quasar 670-labeled probe, respectively. To optimize measurement sensitivity for analysis of highly degraded FFPE samples; a) each RNA sample was reverse transcribed (RT) with gene-specific primer and b) cDNA was pre-amplified with 18 PCR cycles in the presence of internal standards. Results obtained with pre-amp or no pre-amp conditions were highly correspondent. We conducted a validation study of this assay in 38 samples (10 malignant and 10 benign surgical FFPE tissues and 13 malignant and 5 benign fine needle aspirate (FNA) samples. Consistent with previous results in fresh frozen surgical samples, the lung cancer diagnostic test (LCDT) optimal cut-off value discriminated malignant from non-malignant tissues (p = 0.0009), had 92.9% specificity and 75.0% sensitivity, and a receiver operator characteristic area under the curve of 0.87 (95% confidence interval 0.74-0.99). Based on these data, we expect that this quality-controlled two color fluorometric qPCR approach will enable reliable analysis of the LCDT and other promising qPCR-based molecular diagnostic tests in small degraded RNA extracted from clinical FFPE and FNA cell block FFPE samples. Citation Format: Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Laura Jordan, Jill Zyrek, Luis de la Casas, James C. Willey. Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2013-61

Abstract 5538: Development of RNA quality control methods to improve BCR-ABL measurement in whole blood samples

Measurement of BCR-ABL fusion transcripts in whole blood by quantitative, reverse-transcriptase PCR is a clinically validated method to monitor treatment response in patients with chronic myelogenous leukemia. For example, achieving a three-log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A 0.5-log increase from MMR signifies treatment failure sufficient to motivate change in treatment. The purpose of this study was to develop a BCR-ABL test with improved analytical performance characteristics including adequate quality control for RNA degradation or reverse transcription interference and reliable comparison of results across testing sites. To test whether RNA degradation could mask a 0.5-log increase in expression, an RNA degradation model was established by incubating whole blood from each of six individuals with K562 cells at room temperature for various times post-venipuncture. When data from six subjects were combined, the BCR-ABL/103 GUSB value trended down at 24h but was not significantly decreased until 48h (51% decrease, p = 0.004). To quantify the amount of whole blood RNA that can be loaded into an RT reaction without reducing RT efficiency, six different amounts of total RNA extracted from whole blood mixed with K562 cells were added into RT reactions. To measure RT efficiency, an RT Standards Mixture (RTSM) containing known copy numbers of External RNA Control Consortium (ERCC) 171 RNA and ERCC 113 cDNA was added to each RT reaction. RT efficiency was measured as the yield of PCR product from ERCC 171 RNA relative to ERCC 113 cDNA. Based on data from three subjects, compared to RT efficiency at reference concentration of 30 ng/µl RNA in RT, there was a 35% decrease (p = 0.043) at 167 ng/μl RNA and 82% reduction at 1000 ng/μl RNA. The maximum yield of BCR-ABL (molecules/μL cDNA) was observed at 300 ng/µl RNA/RT with a 3-fold increase (p=0.003) compared to 30 ng/µl RNA. Greater than 300 ng/µl RNA/RT did not further increase yield of BCR-ABL molecules/μL cDNA, likely due to reduced RT efficiency. We conclude that BCR-ABL/103 GUSB should be measured within 24 hours following blood collection. Because decreasing RT efficiency is associated with increasing RNA input into RT reactions, including 300 ng/µl RNA in RT reactions provides the maximum sensitivity in measuring BCR-ABL expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5538. doi:1538-7445.AM2012-5538

Abstract 5549: Normal bronchial epithelial cells (NBEC) sample RNA quality characteristics that will yield reliable measurement of lung cancer risk test

Lung cancer is the leading cause of cancer mortality in the United States with cigarette smoking the primary risk factor. Lung cancer has a low survival rate because it typically is at an advanced stage when first detected and treated. In previous studies a Lung Cancer Risk Test (LCRT) was identified comprising transcript abundance measurement of 14 key antioxidant, DNA repair and transcription factor genes measured in normal bronchial epithelial cells (NBEC) sampled after bronchoscopy. The LCRT promises to identify high-risk individuals who will develop lung cancer. This will enable even more focused selection for closer monitoring, further reduce risk of false positive findings and markedly reduce cost of implementation. An ongoing multi-institutional prospective cohort nested case control trial, funded by NCI grant RC2148572 is intended to test the validity of LCRT as an accurate test for lung cancer risk. More than twelve test sites are providing NBEC samples for the LCRT study. Accurate LCRT measurement will be dependent upon sample RNA quality. Thus, the purpose of this study is to identify cut-off criteria according to which NBEC RNA biospecimens can be expected to yield reliable quantitative, reverse-transcriptase polymerase chain reaction (qRT-PCR) results for the genes comprised by the LCRT. RNA quality of each collected NBEC sample was measured using three parameters: quantity, purity and integrity. RNA integrity was measured by one of two tests, GAPD and GUSB. Each test used one reverse primer and two forward primers and a sequence specific fluorescent labeled probe for signal detection. The GAPD test yielded 70bp and 230bp products and the GUSB test yielded 60bp and 120bp products. The integrity of each unknown RNA sample was determined by dividing the yield of longer product by that of shorter product. Of 126 NBEC samples assessed so far, 77.8% provided > 500 ng total RNA, 94.4% had A260/280 values > 1.5 and 95% gave integrity test values > 0.5. To determine the specific cut-off threshold for each NBEC RNA quality criterion above which LCRT results will be reliable, we established an A549 cell line RNA integrity reference model by incubating cell populations at room temperature for different amounts of time following cytolysis, generating samples with varying decreasing integrity. The LCRT will be measured in these intentionally degraded RNA samples, allowing comparison between integrity levels and LCRT values. The integrity level above which measurement of each gene comprised by the LCRT can be expected to be reliable and accurate will be determined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5549. doi:1538-7445.AM2012-5549

Abstract 2232: Effectiveness evaluation of an in vitro nucleic acid amplification test for quantification of BCR-ABL fusion transcript variants in human whole blood

Quantitative measurement of BCR-ABL fusion gene transcripts in whole blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a clinically validated method to monitor tyrosine kinase inhibitor drug response in patients diagnosed with chronic myelogenous leukemia (CML). Monitoring of BCR-ABL expression in blood of cML patients under treatment with a tyrosine kinase inhibitor of BCR-ABL is considered standard of care. A three log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A subsequent 0.5 log increase in BCR-ABL from MMR is considered a sign of treatment failure sufficient to trigger change in treatment. The challenge is to develop a test with sufficient analytical performance characteristics and quality control to reliably measure BCRABL in MMR samples in which there may be less than 10 molecules in 300 of RNA extracted from whole blood. A method validation protocol was developed using the International Committee on Harmonization (ICH) Q2(R1) international validation guidelines. BCRABL variants b2a2 and b3a2 as well as the housekeeping gene GUSB were measured in KCL22 cell line RNA and Stratagene Universal Human Reference RNA test articles in extreme linear dilution assay, robustness testing conditions, and in multiple laboratories. Quality controls were developed to enable loading as much whole blood RNA into the assay as possible without assay interference. These included a reverse transcription (RT) standardization mixture comprising External RNA Control Consortium (ERCC) reagents to control for RT interference, and controls for genomic DNA contamination, RNA integrity, and PCR interference. Results: This test for b2a2 and b3a2 variants of BCR-ABL meets ICHQ2(R1) guidelines for specificity, linearity, accuracy, LOD and LOQ, imprecision, robustness, and reproducibility. Each analyte comprised by the test was linear (R2 > 0.97) over more than 3 logs 10 , and demonstrated inter-experimental and inter-laboratory imprecision low enough to enable measurement of a 3-fold difference as significant (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2011-2232

Abstract 825: Development of RNA quality-control methods to improve tissue archiving and molecular diagnostic testing

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Biospecimen collection and processing methods practiced today yield clinical samples with a wide variation in RNA quality. Efforts to improve these methods are inadequate due to insufficient means to measure RNA quality. Improved RNA quality control (QC) measurement methods will enable establishment of cut-off criteria to determine which biospecimens will yield reliable RT-PCR results. Implementation of such criteria will enable existing banks of samples with variable RNA quality to be used for reverse transcriptase polymerase chain reaction (RT-PCR)-based diagnostic test development. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances that either a) interfere with RT efficiency and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. An RNA integrity reference model was established by incubating A549 cell line populations at room temperature for different amounts of time following cytolysis. Between 0 and 100 min. post cytolysis, ACTB degradation, as well as the degradation of other major housekeeping genes, was more rapid than decline in RNA Integrity Number (RIN) Score, a measurement based on microcapillary electropheresis. Quantification of single-copy CC10 gene gDNA in RNA controlled for gDNA contamination and measurement of transcript abundance of each gene relative to known number of respective cDNA internal standards controlled for PCR inhibitors. The RT inhibition test uses a Reverse Transcription Standards Mixture (RTSM) that contains a known number of copies of ERCC (External RNA Control Consortium) RNA and cDNA standards. A known quantity of RTSM is added to an RNA sample and measurement of PCR product from the RNA standard relative to its cDNA standard following RT-PCR assesses RT efficiency. These RT-PCR tests have been implemented on an Agilent 2100 Bioanalyzer® and are being developed for implementation on high throughput RT-PCR platforms such as the Biotrove OpenArray® nanoarray and the Gene Express Standardized Expression Measurement (SEM) Center™. Improved methods for biospecimen collection will be selected and will then be used to identify which samples collected according to present methods are suitable for analysis by RT-PCR-based molecular diagnostic tests. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 825.