Dawne N. Shelton

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression

Melanoma lacks a clinically useful blood‐based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell‐free, tumor‐associated BRAFmutant and NRASmutant DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAFmutant and NRASmutant ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%–58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non‐RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non‐RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non‐RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction

Background Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Patients and methods Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Conclusion(s) Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.

Abstract 493: Quantity of KRAS mutations in cell-free DNA is associated with survival of patients with advanced cancers

Background: Cell-free (cf) DNA from plasma offers an easily obtainable material for KRAS mutation analysis for diagnostics and monitoring. There is emerging evidence that the percentage of mutant cfDNA in the wild-type background (mutant allele fraction, MAF) and/or absolute quantity of mutant cfDNA can be associated with survival of patients with advanced cancers. Methods: Plasma-derived cfDNA from patients with progressing advanced cancers was purified and 16 ng of DNA was tested with a KRAS multiplex assay to distinguish the wild-type allele from 7 of the most common mutations in the G12 and G13 hotspot of exon 2 using the QX200 Droplet Digital PCR™ platform (Bio-Rad). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory and clinical outcomes including survival. Results: Of the 117 patients (colorectal cancer, 71; non-small cell lung cancer, 12; melanoma, 10; pancreatic cancer, 5; ovarian cancer, 5; appendiceal cancer, 5; other cancers, 9), KRAS mutations were detected in 85 (73%) archival FFPE tumor samples and 85 (73%) plasma cfDNA samples. The two methods had overall agreement in 109 patients (93%; kappa, 0.83, standard error, 0.06; 95% confidence interval [CI], 0.71-0.94), sensitivity of 95% (95% CI, 0.88-0.99), specificity of 88% (95% CI, 0.71-0.96), even though median time from tissue to blood sampling was 18.5 months (1.1-134.4 months). A higher MAF (>7%) of KRAS in cfDNA as determined by 5% trimmed mean value was associated with shorter survival compared to lower ( Conclusions: A higher percentage of KRAS mutation in plasma cfDNA is an independent predictive factor for shorter survival in patients with advanced cancers. Citation Format: Kiran Madwani, Helen J. Huang, Dawne N. Shelton, Siqing Fu, Apostolia M. Tsimberidou, Sarina A. Piha-Paul, Aung Naing, David S. Hong, Daniel D. Karp, Debra L. Andrews, Goran Cabrilo, E. Scott Kopetz, Rajyalakshmi Luthra, Bryan K. Kee, Cathy Eng, Van K. Morris, George A. Karlin-Neumann, Funda Meric-Bernstam, Filip Janku. Quantity of KRAS mutations in cell-free DNA is associated with survival of patients with advanced cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 493.

Abstract 2407: Multiplex KRAS G12/G13 mutation testing of 16ng of unamplified cell-free DNA from plasma of patients with advanced cancers using Droplet Digital PCR

Background: Cell-free (cf) DNA from the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive and repeatedly applicable source of biologic material for mutation analysis of druggable targets and monitoring molecular changes in tumor(s) during and after therapeutic interventions. Novel, multiplex, and accurate diagnostic systems using low amounts of DNA are needed for further development of plasma cfDNA testing in personalized therapy. Methods: cfDNA from plasma samples of patients with advanced cancers who progressed on systemic therapy was purified and 16 ng of DNA was tested with a KRAS multiplex assay to distinguish wild-type allele from 7 of the most common mutations in in the G12/G13 hotspot of exon 2 using the QX200 Droplet Digital PCR™ platform (Bio-Rad, Pleasanton, CA). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory. Results: cfDNA was extracted from plasma samples of 94 patients with advanced cancers (colorectal, n = 60; melanoma, n = 9; non-small cell lung, n = 9; appendiceal, n = 3; ovarian, n = 3; endometrial, n = 3; other cancers, n = 7). KRAS G12/G13 mutations were detected in 62% (58/94) of plasma samples and in 68% (64/94) of archival tumor samples, resulting in concordance in 84 (89%) of patients (kappa = 0.77, 95% confidence interval [CI] 0.63- 0.90) with sensitivity 88% (95% CI 0.77-0.94), specificity 93% (95% CI 0.78-0.99), positive and negative predictive values 97% (95% CI 0.88-0.99) and 78% (95% CI 0.61-0.90), respectively. Overall, 8 patients had KRAS G12/G13 mutation in the tumor, but not in cfDNA and 2 patients had KRAS G12/G13 mutation in cfDNA, but not in the tumor. Of interest, 1 of 2 patients with KRAS G12/G13 mutation (colorectal cancer) in cfDNA, but not in the tumor, experienced rapid disease progression after 1 cycle of cetuximab with chemotherapy. Discrepancies will be addressed with testing of tissue samples using the Bio-Rad QX200 system and repeating cfDNA testing with an increased amount of DNA. Results will be presented at the meeting. Conclusions: Multiplex detecting of KRAS G12/G13 mutations in a low amount of unamplified cfDNA from plasma using the Bio-Rad QX200 platform is a noninvasive alternative to mutation testing of tumor tissue with an acceptable level of concordance and sensitivity, and should be investigated further for testing of KRAS mutation status in patients with cancer. Citation Format: Helen J. Huang, Dawne N. Shelton, Siqing Fu, Sarina A. Piha-Paul, Apostolia M. Tsimberidou, Ralph G. Zinner, Jennifer J. Wheler, Aung Naing, David S. Hong, Gerald S. Falckook, Scott Kopetz, Rajyalakshmi Luthra, Bryan K. Kee, George A. Karlin-Neumann, Funda Meric-Bernstam, Filip Janku. Multiplex KRAS G12/G13 mutation testing of 16ng of unamplified cell-free DNA from plasma of patients with advanced cancers using Droplet Digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2407. doi:10.1158/1538-7445.AM2015-2407

Abstract LB-115: Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR

Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, routine implementation of genomic testing is still limited by: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. In metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor antibodies (αEGFR) are used to target the wild-type EGFR receptor. However, KRAS is mutated in approximately 40% of colorectal cancers and is indicative of a negative response to αEGFR therapy. BRAF and PIK3CA mutations are also associated with poor response in the remaining patients with KRAS wild-type genotype. To optimize therapy strategies for personalized care, it is therefore critical to rapidly screen patient samples during the course of disease for the presence of multiple mutations. The low abundance of mutants and limited amount and quality of available clinical samples (FFPE and cfDNA) render it difficult to reliably detect multiple mutations with current platforms and methods. We have developed a multiplexing strategy for screening clinically-actionable KRAS, BRAF, and PIK3CA mutations in mCRC clinical samples using digital PCR. No pre-amplification step was required. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented in molecular diagnostic laboratories for rapid, routine screening and monitoring of residual disease in cancer patients. Citation Format: Wei Yang, Dawne N. Shelton, Samantha Cooper, Jennifer Berman, Svilen Tzonev, Eli Hefner, John F. Regan. Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2014-LB-115

Abstract 2301: Cross validation of NGS methylated targets using droplet digital PCR (ddPCR)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA methylation is one of the most studied epigenetic modifications. DNA methylation plays an important role in a number of physiological processes as well as common diseases such as cancer and neurodegenerative disorders. In mammals, DNA methylation occurs at the C-5 position of cytosine in CpG dinucle¬otide sequences, which are mainly concentrated in regions known as CpG islands. Methylation in CpG islands within gene promoters usually leads to gene silencing. Recent data have shown the correlation of methylation and disease status. However, there are significant limitations in both biology and technology. Biologically, methylation status is highly heterogeneous in nature. In normal controls there is never a complete lack of methylation marks, and conversely, never a completely saturated methylation signature. In addition methylation sites of interest are often located in CpG islands, regions where multiple methylation sites occur side by side, making assay design and discrimination challenging. Technological hurdles add significant variability as well. The typical method for determining methylation is bisulfite conversion, which uses harsh chemicals to convert cytosines to uracil. When a cytosine is methylated, it is largely, though not completely, protected from the conversion process. However, bisulfite conversion also causes a great deal of DNA damage, causing the strands to separate and fragment. In most cases of research, the DNAs that researchers want to examine are from FFPE or cfDNA samples, samples which are already both limited and damaged. Because of these challenges, there is an unmet need for a sensitive yet robust method to analyze DNA methylation in clinical samples in a rapid, inexpensive, and high throughput manner. Herein we showcase the use of ddPCR for detection of DNA methylation in bisulfite converted gDNA samples. First we demonstrate the superior sensitivity, linearity and robustness of ddPCR methylation detection in the SNRPN promoter, a model system for an imprinted gene involved in neurological disorders. Secondly we show the detection of DNA methylation in the vimentin promoter, which is known to be methylated in cancer (Li et al. Nat. Biotech. 2009). We will demonstrate reproducible detection in replicate bisulfite converted samples and sensitivity. Citation Format: Dawne N. Shelton, Claudia Litterst, John F. Regan, Helen R. Moinova, Sanford D. Markowitz. Cross validation of NGS methylated targets using droplet digital PCR (ddPCR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2014-2301