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Purpose: To evaluate the detection of gene mutations in bone marrow biopsy and circulating free DNA (cfDNA) from plasma in multiple myeloma (MM). Experimental design: We used cell-free DNA from plasma and bone marrow to test BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (ddPCR), and evaluated results with clinical outcomes. Results: We found of 83 patients, the detectable mutation frequencies for the above four genes were 4 (5%), 13 (16%), 3 (4%) and 14 (17%) in bone marrow, respectively. The median variant allelic frequency (VAF) in most mutations were 1.595%. In 17 paired cfDNA samples, the detectable mutation frequencies for the above four genes were 5 (30%), 1 (6%), 0 (0%) and 3 (18%) respectively, and the median VAF rate was 2.9%. Agreement between bone marrow DNA and plasma cfDNA were 76%, 100%, 100% and 100% compared to the tissue detections, respectively. In 17 patients with paired bone marrow and plasma samples, the above four mutations were 3 (18%), 1 (6%), 0 (0%) and 2 (12%) respectively, with the agreement rates of 88%, 88%, 100% and 100% compared to tissue detections. Of 57 patients with available outcome data, high mutation VAF had a shorter median survival than patients with low mutation VAF (P=0.0322). Conclusions: Oncogenic mutations in BRAF, KRAS and NRAS genes can be detected in the bone marrow and plasma cfDNA with ddPCR in patients with MM patients and high VAF is associated with short survival.

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERT(mutant) DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERT(mutant) tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERT(mutant) DNA in tumors and plasma of cancer patients.

Despite the interest in characterizing all genomic variation, the presence of large repeats at the breakpoints of many structural variants hinders their analysis. This is especially problematic in the case of inversions, since they are balanced changes without gain or loss of DNA. Here we tested novel linkage-based droplet digital PCR (ddPCR) assays on 20 inversions ranging from 3.1 to 742 kb and flanked by long inverted repeats (IRs) of up to 134 kb. Among these, we validated 13 inversions predicted by different genome-wide techniques. In addition, we have generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions for which there were no high-throughput methods to determine directly the orientation, like the well-characterized 17q21 inversion. Through comparison with previous data, independent replicates and both inversion breakpoints, we have demonstrated that the technique is highly accurate and reproducible. Most of the studied inversions are frequent and widespread across continents, showing a negative correlation with genetic length. Moreover, all except two show clear signs of being recurrent, and the new data allowed us to define more clearly the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able to check inversion functional effects in multiple tissues, validating gene expression differences reported before for two inversions and finding new candidate associations. Our work therefore provides a tool to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT–124C>T, and TERT–146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT–124C>T and TERT–146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Introduction: Serial monitoring of sensitive and specific blood-based biomarkers of disease burden in cancer patients could enable clinicians to adjust treatments at the earliest signs of disease progression when the likelihood of improved outcomes is probably greatest. Such assays require preanalytical, analytical, and clinical validation prior to use. We conducted analytical validation studies of 7 droplet digital PCR (ddPCR) assays to determine their performance characteristics in detecting mutant TERT, BRAF, and NRAS ctDNA. Methods: Probe-based ddPCR assays (Bio-Rad) detecting BRAF V600E, K, NRAS Q61K, R, L, and TERT C228T, C250T were studied. Preanalytical validation experiments using plasma collected in EDTA tubes investigated processing variables including plasma volumes, centrifugation speeds, and DNA extraction efficiencies, and evaluated the necessity of internal controls using spike-in experiments. Analytical validation studies assessed accuracy using plasma from healthy donors spiked with known amounts of mutant nucleosomal DNAs from control cell lines. Potentially interfering substances were evaluated using the AcroMetrix inhibition panel (Thermo Scientific). Precision/reproducibility was assessed using combinations of multiple reagent lots and operators. Assay specificity was determined using templates with different mutations to check cross-reactivity. Limits of Blank (LoB) and Limits of Detection (LoD) were calculated for each assay using 8 replicate wells of healthy donor plasma or mutant cell line DNA, respectively. Three-phase inter-laboratory harmonization was conducted using: 1) extracted DNA, 2) plasmas spiked with control DNAs, and 3) melanoma patient samples shipped in PAXgene Blood ccfDNA tubes (Qiagen). Results: Preanalytical validation yielded a double centrifugation protocol to reduce contaminating large DNA. Assays were highly accurate and precise with 80-90% recovery of spiked-in mutant templates and coefficients of variation (CVs) Conclusion: This ddPCR-based plasma analysis platform demonstrates low background levels in normal plasma, yielding high sensitivities to detect small amounts of ctDNAs with these mutations. The assays also demonstrate highly reproducible results between laboratories. These assays could be integrated into biomarker-driven clinical trials to evaluate their potential to inform more personalized clinical decision-making. Citation Format: Mahrukh M. Syeda, Broderick Corless, Iman Osman, Cindy Spittle, George Karlin-Neumann, David Polsky. Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A66.

Introduction: Liquid biopsies, especially plasma cell-free circulating tumor DNA (ctDNA), provide a potential opportunity to be a noninvasive biomarker for the diagnosis and monitoring of glioblastoma (GBM) patients. Previously, we detected TERT promoter hotspot mutations (C228T and C250T) in ctDNA of IDH wild-type (IDHwt) TERT promoter mutant GBM patients with 100% specificity using mutation-specific droplet digital PCR (ddPCR) assays. Here, we examine the association between mutant TERT ctDNA levels and clinical outcomes in newly diagnosed GBM patients undergoing chemoradiation. Methods: We analyzed 76 serially collected plasma samples from 17 patients with suspected IDHwt GBM based on MRI before surgery. Twenty mL of whole blood was collected in EDTA tubes at predetermined times: pre- and postoperatively, at the end of chemoradiation, and 1, 3, and 6 months from the end of chemoradiation. TERT promoter mutations C228T or C250T were identified in FFPE tumor samples using ddPCR assays specific for these mutations. Plasma samples were analyzed for the patient’s tumor TERT mutation using the ddPCR assays. The analytically validated thresholds for positive ctDNA detection were 1.5 and 1.7 copies/mL for C228T and C250T, respectively. Results: Sixteen of 17 (94%) IDHwt tumors had TERT mutations (10 C228T, 6 C250T) with MGMT methylated, unmethylated, or unknown status in 10, 5, and 1, respectively. Fourteen of the 16 patients (87.5%) had detectable mutant ctDNA at one or more time points (range 1.66 to 22.13 copies/mL). Of the 2 patients with undetectable ctDNA, one had diffuse and non-avidly enhancing disease and the other only had pre/postop plasma samples collected. Six patients had detectable ctDNA preop, and most had a dominant rim-enhancing mass with additional nonenhancing or enhancing lesion(s). Ten patients had detectable ctDNA up to 4 days postop, half of whom had undergone gross total resection. For 3 of 5 patients for whom there was a question of pseudoprogression versus true progression, ctDNA kinetics matched the clinical outcome. One patient with MGMT unmethylated multifocal GBM achieved ctDNA zeroconversion at 6 months post radiation (RT), and did not progress for another five months. Another patient was negative at all time points until their 3-month post RT follow-up, at which time they developed a recurrence. Another patient achieved zeroconversion at the end of RT but developed a borderline positive ctDNA at 6 months after RT, 2 months before documented radiographic progression. Conclusions: In this pilot, prospective ctDNA monitoring study of IDHwt GBM, TERT mutant ctDNA was detected at one or more time points in the majority of patients. ctDNA kinetics were associated with clinical outcomes for some patients. These data suggest that additional, larger studies could refine how ctDNA monitoring may be used to enhance the clinical management of IDHwt GBM patients. Citation Format: Christine Cordova, Mahrukh M. Syeda, Broderick Corless, Jennifer M. Wiggins, Amie Patel, Sylvia C. Kurz, Malcolm Delara, Zacharia Sawaged, Minerva Utate, Dimitris Placantonakis, John Golfinos, Jessica Schafrick, Joshua S. Silverman, Rajan Jain, Matija Snuderl, David Zagzag, George Karlin-Neumann, David Polsky, Andrew S. Chi. Longitudinal detection of TERT-mutant plasma cell-free circulating tumor DNA in newly diagnosed glioblastoma patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A65.

Besides quantifying the absolute number of copies of known DNA targets, digital PCR can also be used to assess whether two nonpolymorphic gene sequences or two heterozygous markers reside on the same DNA molecule (i.e., are physically linked). Some useful linkage applications include: phasing variants to define a haplotype; genotyping of inversions; determining the presence of multimarker pathogenic bacteria in a metagenomic sample; and assessing DNA integrity. This chapter describes an efficient and cost-effective method for analyzing linkage of any two genetic sequences up to at least 200 Kb apart, including phasing of heterozygous markers such as that which occur abundantly in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

After several decades of relatively modest use, in the last several years digital PCR (dPCR) has grown to become the new gold standard for nucleic acid quantification. This coincides with the commercial availability of scalable, affordable, and reproducible droplet-based dPCR platforms in the past five years and has led to its rapid dissemination into diverse research fields and testing applications. Among these, it has been adopted most vigorously into clinical oncology where it is beginning to be used for plasma genotyping in cancer patients undergoing treatment. Additionally, innovation across the scientific community has extended the benefits of reaction partitioning beyond DNA and RNA quantification alone, and demonstrated its usefulness in evaluating DNA size and integrity, the physical linkage of colocalized markers, levels of enzyme activity and specific cation concentrations in a sample, and more. As dPCR technology gains in popularity and breadth, its power and simplicity can often be taken for granted; thus, the reader is reminded that due diligence must be exercised in order to make claims not only of precision but also of accuracy in their measurements.

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes. This chapter presents a quick and efficient 1-day workflow for sorting single cells with a small laboratory cell-sorter followed by an ultrahigh sensitivity, multiplexed digital PCR method for quantitative tracking of changes in 5-10 genes per single cell.

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 BRAF(mutant) and NRAS(mutant) 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 BRAF(mutant) and NRAS(mutant) 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

Besides quantifying the absolute number of copies of known DNA targets, digital PCR can also be used to assess whether two nonpolymorphic gene sequences or two heterozygous markers reside on the same DNA molecule (i.e., are physically linked). Some useful linkage applications include: phasing variants to define a haplotype; genotyping of inversions; determining the presence of multimarker pathogenic bacteria in a metagenomic sample; and assessing DNA integrity. This chapter describes an efficient and cost-effective method for analyzing linkage of any two genetic sequences up to at least 200 Kb apart, including phasing of heterozygous markers such as that which occur abundantly in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes. This chapter presents a quick and efficient 1-day workflow for sorting single cells with a small laboratory cell-sorter followed by an ultrahigh sensitivity, multiplexed digital PCR method for quantitative tracking of changes in 5-10 genes per single cell.

Dear Editor, We wish to raise some questions and concerns about the design, execution and evaluation of results in Sherwood et al .1 Concerning the study design, it is not clear that the same amount of sample material was analysed by all of the technologies tested. For example, while it appears that most platforms processed 1 μL of each sample in table 2 (ie, 50 or 100 mutant copies per test reaction), it seems that for Oncomine duplicate libraries were made and their results averaged in table 3, potentially enhancing the assessed sensitivity of this platform. Was data for all other platforms derived from a single experimental run of the sample test plate? For droplet digital PCR (ddPCR) tests, was 9 μL of each of these undiluted samples …

Purpose: The mutational spectra of melanoma has been well characterized; however, the presence of distinct subclones among multiple tumors from a given patient has been less well described. As mutational heterogeneity has been associated with decreased responses to treatments in other cancers, we sought to estimate the occurrence of distinct subclones within individual melanoma patients by analyzing commonly mutated melanoma genes using a multi-platform mutation-detection approach. Methods: We analyzed 271 formalin-fixed, paraffin embedded tumors from 99 patients with stage III or IV melanoma enrolled in the NYU Melanoma Biorepository. All patients had two or more available tumor specimens, and complete clinical data. All samples were reviewed for adequate tumor content, and extracted DNA was assessed for mutations at BRAFV600, NRASQ61, and TERT-124C>T and TERT-146C>T using a combination of multiplex SNaPshot assays, Sanger Sequencing, Allele-specific real-time PCR, or droplet digital PCR (ddPCR). Samples undergoing ddPCR analysis for TERT mutations were treated with uracil-DNA glycolase prior to amplification to remove C>T artifacts from formalin-fixation. Results: Sixty patients had a primary plus one or more metastatic tumors available; 39 patients had multiple metastatic tumors, but no primary tumor available. Overall, 88% of patients had tumors with at least one BRAF, NRAS and/or TERT mutation. We identified inter-tumor mutational heterogeneity in 20/99 (20%) patients, with TERT mutational heterogeneity present in 15 of these patients. Among 14 patients with heterogeneity between their primary and metastatic tumors, 6/14 (43%) had additional mutations in their metastases compared to their primaries. Most interestingly, 8/14 (57%) patients had mutations in their primary that were undetectable in at least one of their metastases; 5 of these patients had TERT mutational heterogeneity. Three patients had a BRAF mutation in their primary that was undetectable in at least 1 of their metastases. One patient had a BRAFV600E/NRASWILD-TYPE/TERTWILD-TYPE primary on the leg and 3 regional metastases lacking BRAF mutations; but carrying NRASQ61K and TERT-124C>T mutations. Another patient had 3 different metastatic tumors, with 3 different mutational spectra. We did not detect any tumors with simultaneous BRAF and NRAS mutations; however, we did detect both TERT-124C>T and TERT-146C>T mutations in 7 tumors from 7 individual patients. Four of these were primary tumors, and metastases from these patients lacked 1 of the 2 TERT mutations identified in the primary. Conclusion: Clonal heterogeneity in melanoma is fairly common as evidenced by divergent detection of TERT, BRAF and NRAS mutations using high sensitivity multi-platform mutation detection analyses of multiple melanoma tumors from individual patients. Heterogeneity appears to occur in primary tumors. Citation Format: Gregory Chang, Broderick Corless, Nathaniel Fleming, Cindy Spittle, Farbod Darvishian, Anna Pavlick, Russell Berman, Richard Shapiro, George Karlin-Neumann, Iman Osman, David Polsky. Identification of melanoma mutational tumor heterogeneity using BRAF, NRAS and TERT-promoter mutation-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4704.

Purpose: Challenges to accurate quantitative measurements of circulating tumor DNA (ctDNA) include variable contamination with large DNA fragments released during mononuclear cell lysis, and potential variability in extraction efficiency. We investigated optimal procedures for plasma processing and the potential utility of a DNA spike-in as a process control for extraction efficiency. Methods: Initial experiments analyzed the potential benefits of different centrifugation protocols (1600 x g vs. 1600 x g followed by 16000 x g) for plasma separation by analyzing the size distribution of extracted DNA using a BioAnalyzer. In subsequent experiments we used droplet digital PCR (ddPCR) to compare differences in extraction efficiency between nucleosomal DNA (nDNA) and genomic DNA (gDNA) spiked into healthy donor plasma and extracted using the QIAamp DSP Circulating NA Kit (Qiagen). To evaluate the properties of the purification columns with respect to the yield of different DNA sizes, we compared the plasma results to experiments extracting purified nDNA and gDNA spiked into PBS instead of plasma. We investigated the potential competitive effect of large DNA on the extraction efficiency of small DNA by mixing nDNA with commercially available human gDNA in different concentrations. To assess if nDNA, when spiked into patient plasma, would track with endogenous ctDNA, we spiked a pool of plasma from patients with BRAFV600E mutations with known quantities of nDNA purified from NRASQ61K mutant cells (CHP212). The spiked plasma pool was divided and extracted using multiple separate columns. Copies/ml and mutant fraction were determined by ddPCR assays for BRAFV600E and NRASQ61K. Coefficients of variation were also calculated. Results: Comparison of different centrifugation speeds for plasma processing before and after freeze-thaw indicated that a 2nd high-speed centrifugation (16000 x g), either before or after a freeze-thaw cycle, minimizes large DNA in plasma samples prior to ctDNA extraction. In initial spike-in experiments extraction efficiency for nDNA was 87.7% compared to 42% for gDNA. We obtained similar results when purifying the nDNA and gDNA from PBS instead of plasma. We observed greater variability in nDNA extraction efficiency when increasing amounts of gDNA were added as a contaminant. Finally, we found that the slight, paralell variations in extraction yields of externally spiked NRASQ61K mutant nDNA and endogenous BRAFV600E ctDNA in 7/9 replicate extractions. The overall coefficients of variation (total copies/mL) for 9 extractions for BRAFV600E and NRASQ61K were 3.9 and 4.2 respectively. Conclusion: A second centrifugation step helps reduce potential contamination of ctDNA with large DNA, and may improve the recovery of ctDNA from plasma samples. Nucleosomal DNA can potentially be used as a process control for extracting ctDNA from plasma samples. Citation Format: Mahrukh M. Syeda, George Karlin-Neumann, Iman Osman, David Polsky. Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2239.

2026 Background: There is a critical need for more specific and less invasive diagnostic and pharmacodynamic biomarkers in glioblastoma (GBM) patients (pts). Previously, we detected TERT promoter hotspot mutations (C228T and C250T) in the ctDNA of IDH wildtype ( IDHwt) TERT promoter mutant GBM pts with 100% specificity using mutation-specific droplet digital PCR (ddPCR) assays. Here, we explored the dynamics and clinical associations of mutant TERT ctDNA levels in GBM pts undergoing therapy. Methods: We examined 14 pts with suspected IDHwt GBM based on preoperative MRI. Plasma was isolated and frozen from ~15 mL whole blood samples collected pre- and post-op, at end of radiation (RT), and 1, 3, and 6 m after end of RT. TERT promoter mutations were identified in FFPE tumor samples using ddPCR assays for C228T/C250T. Plasma samples were analyzed using ddPCR assays specific for the corresponding tumor mutation. The validated thresholds for positive detection were 1.5 (C228T) and 1.7 copies/mL (C250T). Results: 13/14 (92.9%) IDHwt tumors had TERT mutations (7 C228T and 6 C250T). Six of these 13 (46%) pts had positive plasma TERT ctDNA preop (4 C228T, 2 C250T). The mean cross sectional area of enhancing disease at presentation for positive or negative preop mutant ctDNA was similar. All 4 pts with multiple contrast enhancing lesions had positive preop mutant ctDNA. 2 pts who were negative initially developed detectable mutant ctDNA preceding progression. 3/4 pts with equivocal radiographic pseudoprogression had ctDNA dynamics that correlated with eventual clinical outcome. One patient with unresectable GBM had declining mutant ctDNA in later collections during clinical stability. Conclusions: We detected plasma TERT ctDNA in 46% of TERT mutant GBM pts before surgery, and in 100% of pts with multiple contrast enhancing lesions. TERT mutant ctDNA levels correlated with pseudoprogression or true disease progression and predicted progression before MRI. These data suggest that larger studies to test circulating cell-free TERT mutation as a diagnostic and pharmacodynamic biomarker in GBM are warranted.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

Background Cell-free DNA (cfDNA) from plasma offers easily obtainable material forKRAS 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 aKRASG12/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%) hadKRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) hadKRASG12/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% ofKRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P=0.001). Conclusion(s) Multiplex detection ofKRASG12/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 mutantKRASG12/G13 in cfDNA corresponded with shorter survival.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies. Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw. Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget. Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge. Citation Format: Broderick Corless, Greg Chang, Weihua Liu, Jin Li, Andre Marziali, Laura Mai, Matthew Wiggin, Melissa Wilson, Anna Pavlick, Iman Osman, George Karlin-Neumann, Cindy Spittle, David Polsky. Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAFmutant metastatic melanoma undergoing treatment with BRAF-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5531.

Background: Oncogenic mutations in BRAF, KRAS and NRAS genes have been reported in multiple myeloma and can be explored as biomarkers for selection of targeted therapies. Methods: Unamplified DNA from archival tumor tissue samples and cell-free DNA (cfDNA) from serum and bone marrow aspirates from patients with multiple myeloma was tested for BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (Bio-Rad). Agreement among methods was evaluated and results were compared to clinical outcomes. Results: Of 88 patients with multiple myeloma (stage I, 13 [15%]; stage II, 26 [30%]; stage III, 34 [39%]; stage unknown, 15 [17%]), 4 (5%) patients were found to have BRAF V600 mutation (all confirmed V600E), 15 (17%) KRAS G12/G13 mutation, 3 (3%) NRAS G12/G13 mutation and 16 (18%) NRAS Q61 mutation in archival tumor tissue. Most mutations were subclonal with a median variant allelic frequency (VAF) 1.55% (range, 0.13%-34%). Survival data were available for 47 patients and patients with high mutation VAF had a trend to shorter survival than patients with low mutation VAF (25.3 months vs. not reached, P=0.087). Of 88 patients, 22 (25%) had simultaneous serum cfDNA collection, which demonstrated BRAF V600 mutation in 7 (32%; 3 confirmed as V600E), KRAS G12/G13 mutation in 2 (9%), NRAS G12/G13 mutation in 0 (0%) and NRAS Q61 mutation in 4/22 (18%) of samples and median VAF was 2.9% (range, 0.34%-18.8%). Agreement rates for serum compared to tissue were 73% for BRAF V600, 95% for KRAS G12/G13, 100% for NRAS G12/G13 and 95% for NRAS Q61. In addition, 13 (15%) patients had simultaneous bone marrow aspirate cfDNA collection, which demonstrated BRAF V600 mutation in 1 (8%; confirmed as V600E), KRAS G12/G13 mutation in 0 (0%), NRAS G12/G13 mutation in 0 (0%) and NRAS Q61 mutation in 1 (8%) of samples. Agreement rates for bone marrow aspirates compared to tissue were 100% for BRAF V600, 92% for KRAS G12/G13, 100% for NRAS G12/G13 and 100% for NRAS Q61. Conclusion: Common oncogenic mutations in BRAF, KRAS and NRAS genes are prevalent with relatively low VAF in multiple myeloma and can be detected with sensitive techniques such as ddPCR. Their prognostic significance and therapeutic utility needs to be further investigated. Citation Format: Filip Janku, Qian Li, Helen J. Huang, Yafei Wang, Zeng Cao, George Karlin-Neumann, Zhiqiang Liu. BRAF, KRAS, and NRAS mutations in archival tumor samples and samples of cell-free DNA from serum and bone marrow aspirates from patients with multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 573.

Purpose: Our goal is to develop response predictive biomarkers for melanoma patients that are more sensitive than standard measures of disease activity. TERT promoter mutations occur frequently in many cancers, especially melanoma. We developed droplet digital PCR assays that detect the 2 most frequently occurring TERT promoter mutations, C228T and C250T. We previously demonstrated that these assays could detect TERTmutant cell-free, circulating tumor DNA (ctDNA) in the plasma of metastatic melanoma patients. In the current study we sought to compare the clinical sensitivity and specificity of these blood-based assays to the current blood biomarker, lactate dehydrogenase (LDH), in detecting the presence of metastatic melanoma and disease progression among patients with unresectable Stage III/IV disease. Methods: We analyzed 71 patients' tumors with unresectable stage III/IV metastatic melanoma undergoing treatment with immuno-oncology therapies, prospectively acquired by the NYU Interdisciplinary Melanoma Cooperative Group. TERT mutations were identified in tumors using droplet digital PCR (ddPCR) or SNaPShot assays (prior to the development of the ddPCR assays). Patients with TERT mutant tumors were selected for further study based on the availability of serial blood collections and radiographic scan data. ddPCR assays used to analyze ctDNA from these patients were chosen based on the tumor mutation detected. Plasmas used for ctDNA analysis ranged from 1-5 mL (73% having at least 4 mL). The ctDNA results were compared with LDH levels and disease status based on radiographic scans and clinical notes. Results: We identified TERT mutations in 31/71 (44%) tumors (18/31 C228T and 13/31 C250T). Our final data set consisted of 26 patients with 85 plasmas. Among patients with available pretreatment samples, ctDNA and LDH were elevated in 14/24 (58%) and 5/24 (21%) patients, respectively (p-value=0.012, McNemar exact test). Among patients whose disease progressed, ctDNA and LDH were elevated at the time of progression in 11/16 (69%) and 4/16 (25%) patients, respectively (p=0.039). Among the patients who had either a complete response (n=2), partial response (n=3), or stable disease (n=3), 1/8 had a slightly elevated ctDNA (3.8 copies/mL with normal level Conclusion: These data demonstrate that ddPCR assays detecting TERT C228T or C250T mutations in ctDNA of metastatic melanoma patients have a greater sensitivity to detect the presence of metastatic disease and disease progression than the currently used standard, LDH. Citation Format: Mahrukh M. Syeda, Broderick Corless, Melissa Wilson, Yesung Lee, Jeremy Tchack, Todd Wechter, Una Moran, George Karlin-Neumann, Anna C. Pavlick, Iman Osman, Yongzhao Shao, David Polsky. Analysis of TERTmutant circulating tumor DNA as a potential biomarker of disease activity in patients with unresectable stage III/IV melanoma receiving immuno-oncology therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5534.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Purpose: Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. Mutations in 1 of 2 hot spots in the TERT promoter sequence are found in several cancers, including up to 85% of melanomas and the majority of cases that lack BRAF or NRAS mutations (about one-third of melanomas). Due to the high G-C content of the TERT promoter sequence these mutations can be difficult to detect using NGS approaches. We developed novel droplet digital PCR (ddPCR) assays to detect these 2 mutations with high sensitivity and specificity, and demonstrate the application of these assays in melanoma clinical samples. Methods: Assays were optimized using cell lines with Sanger sequencing-confirmed mutations: glioblastoma A172 (C228T), and melanoma NYU12-126 (C250T). We varied assay designs and amplification conditions to optimize probe-based detection using the Bio-Rad QX-200 ddPCR system. Assay sensitivities and specificities at various DNA input levels were determined using serial dilutions with 3 replicate wells for each condition. Sensitivity is defined as the lowest mutant allele dilution for which the confidence interval did not overlap with that of the 0% mutant wells. We used normal and cancer-derived DNA sources of different quality (e.g. normal human DNA (Promega), cancer cell lines, plasma and FFPE-derived DNAs) with and without the mutations, and compared the efficiency of detection of amplicons of 88, 113 and 163 base-pairs. We compared efficiencies to assays of similar size for RPP30, a housekeeping gene. Patient-matched metastatic melanoma tumors and plasma samples were analyzed to explore the clinical utility of these assays. Results: The assays showed greater sensitivity when higher amounts of DNA were analyzed. For C228T the limit of detection (LOD) of the mutant allele was 1%, 0.25% and 0.1% for 6.6ng/well, 33ng/well and 66ng/well respectively; for C250T the LODs were 0.25%, 0.05% and 0.05% respectively. Using normal human DNA, the efficiency of the TERT assays averaged approximately 90% of that for RPP30 across assays of similar size, and no decrease in assay efficiency was observed as amplicon length increased. In contrast, whereas amplicon size had only a modest effect on assay efficiency in plasma cfDNA, it gave a more pronounced effect on FFPE DNA’s, decreasing to 38% for the 163bp amplicon. We observed 100% concordance between TERT mutation detection by SNaPshot and ddPCR in 10 FFPE tumor samples, and in plasma samples from 4 metastatic melanoma patients with matching tumor samples. Conclusion: We developed robust ddPCR assays to detect TERT promoter mutations with high sensitivity and specificity. Mutated TERT DNA can be detected and quantitated in the plasma of patients with metastatic melanoma, and is likely to be present in the plasma of other cancer patients in whom TERT mutations occur. Citation Format: Broderick Corless, Gregory Chang, Samantha Cooper, Mahrukh Syeda, Iman Osman, George Karlin-Neumann, David Polsky. Detection of TERT C228T and C250T promoter mutations in melanoma tumor and plasma samples using novel mutation-specific droplet digital PCR assays [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 743. doi:10.1158/1538-7445.AM2017-743

Background: Cell-free (cf) DNA from plasma of patients with advanced cancers offers an easily obtainable material for detection of IDH mutations, which can be used for diagnostics and monitoring purposes. Methods: Plasma was serially collected from patients with advanced cancers with IDH1 R132 or IDH2 R172 mutations, who were enrolled to clinical trials with IDH1, IDH2 or pan-IDH inhibitors. cfDNA was purified and up to 16 ng of each DNA was tested for mutations in the R132 hotspot of IDH1 and R172 hotspot of IDH2 using the QX200 Droplet Digital PCR™ platform (Bio-Rad). Results (mutation allele frequency [MAF] in the wild-type background or the number of IDH-mutant copies/mL of plasma) were compared both to treatment outcomes and to the results of mutation analysis of either archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory. Results: Of the 28 patients (cholangiocarcinoma, 24; other cancers, 4) with IDH1 R132 mutations (N=25) or IDH2 R172 mutations (N=3) in the tumor tissue, IDH mutations were detected in 26 (93%) of plasma cfDNA samples even though median time from tissue to plasma sampling was 16.5 months (3.5-71.1 months). Quantity (< median vs. > median MAF or copies/mL) of IDH-mutant cfDNA was not associated with survival (median not reached vs. 11.1 months, P=0.26). The best radiological response to treatment with IDH inhibitors was stable disease for > 4 months (5/28, 18%) and the median progression-free survival was 1.8 months (95% CI 1.4-2.2). The median quantity of IDH-mutant cfDNA (MAF and copies/mL) at baseline, on therapy and at disease progression did not differ (P=0.16). Changes in quantity (MAF and copies/mL) of IDH-mutant cfDNA within first 3 weeks of therapy were not associated with response on radiographic imaging (P>0.65) or progression-free survival (P>0.12). Conclusions: Detection of IDH mutations in a small amount of unamplified plasma cfDNA using ddPCR has excellent sensitivity compared to conventional clinical IDH mutation testing of archival specimens. Quantity and change in IDH-mutant cfDNA did not correspond with treatment outcomes. Citation Format: Filip Janku, Helen J. Huang, Rachna T. Shroff, Milind Javle, Anthony P. Conley, Anjali Raina, Veronica R. Holley, Aung Naing, Daniel D. Karp, David Fogelman, Ahmed O. Kaseb, Rajyalakshmi Luthra, George A. Karlin-Neumann, Funda Meric-Bernstam. Detection and monitoring of IDH mutations in unamplified plasma cell-free DNA in patients with advanced cancers treated with IDH inhibitors [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 5687. doi:10.1158/1538-7445.AM2017-5687

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.

Highly multiplexed genomics assays are challenged by the need for a sufficient signal-to-noise ratio for each marker scored on a microarray-detection platform. Typically, as the number of markers scored (or target complexity) increases, either more assay-target material must be applied to the array or the specific activity of each marker must be proportionately increased. However, hybridization of excessive amounts of target to the microarray can result in elevated nonspecific binding and consequent degradation of information. We have found that quantum dots provide a successful alternative to organic dyes for achieving highly multiplexed (20,000-plex) and highly accurate, four-color genotyping and have the additional advantage of being excitable by a single wavelength of light despite their distinct emission wavelengths.

Highly multiplexed genomics assays are challenged by the need for a sufficient signal-to-noise ratio for each marker scored on a microarray-detection platform. Typically, as the number of markers scored (or target complexity) increases, either more assay-target material must be applied to the array or the specific activity of each marker must be proportionately increased. However, hybridization of excessive amounts of target to the microarray can result in elevated nonspecific binding and consequent degradation of information. We have found that quantum dots provide a successful alternative to organic dyes for achieving highly multiplexed (>20,000-plex) and highly accurate, four-color genotyping and have the additional advantage of being excitable by a single wavelength of light despite their distinct emission wavelengths.

A new protocol for using molecular inversion probes to specifically and accurately measure allele copy numbers., We have developed a new protocol for using molecular inversion probes to accurately and specifically measure allele copy number. The new protocol provides for significant improvements, including the reduction of input DNA (from 2 μg) by more than 25-fold (to 75 ng total genomic DNA), higher overall precision resulting in one order of magnitude lower false positive rate, and greater dynamic range with accurate absolute copy number up to 60 copies.

Complex human diseases are known to have a significant genetic component. Despite some important successes (Altshuler et al. 2000; Hugot et al. 2001), the elucidation of the underlying genetic determinants have proven resistant to standard methods. Linkage analysis using affected sib pairs has limited power to uncover such signals, as each individual functional variant contributes only modestly to disease risk (Risch 2000). Large-scale association studies that would allow genome-wide mapping in large collections of cases with matched controls have therefore been proposed (e.g., Kruglyak 1999; Risch 2000). Some of the barriers to adopting such strategies, including the need to establish large-case control populations (Geschwind et al. 2001; Shmulewitz et al. 2001; Cupples et al. 2003) and develop comprehensive SNP resources (dbSNP), have been overcome. In addition, the International HapMap project (The International HapMap Consortium 2003) is completing a first draft of a whole genome haplotype map in the Centre d'Etude du Polymorphisme Humain (CEU) population by the end of 2004. The Hap-Map effort will yield a broad view of the genetic architecture of human populations and allow for the efficient selection of the most informative tagging SNPs for subsequent association studies. The remaining requirement to fully enable large-scale genetic association studies is the development of truly cost-effective and scalable SNP genotyping technologies. These methods must allow hundreds of thousands of markers to be efficiently and accurately scored in thousands of patients. The first generation of SNP genotyping technologies were based on single amplification reactions for each locus and were not appropriate for these largescale, whole-genome studies. Recent advances have assayed thousands of random SNPs using ultra high-density wafer hybridizations (Matsuzaki et al. 2004) but will not allow the advantages of the tagging SNP approach to be fully realized because these technologies will not be able to convert all the informative tagging SNPs and/or putative functional SNPs into working genotyping assays. Multiplexed genotyping technologies that allow the multiplexing of hundreds of targeted SNPs have recently been developed based on various versions of the Oligo Ligation Assay (OLA) (Grossman et al. 1994; Samiotaki et al. 1994; Oliphant et al. 2002). These technologies would still demand a fairly large infrastructure in order to process the thousands of reactions necessary per patient. Here, we describe an advanced Molecular Inversion Probe (MIP) genotyping technology (Hardenbol et al. 2003) that combines the strengths of all of the above methods. MIP exploits the advantages of the OLA methodologies but can be more highly multiplexed due to a unique unimolecular method of action and an enzymology that combines the specificity of both ligase and polymerase enzymes. This assay enables the use of >12,000 oligo probes to simultaneously interrogate human genomic DNA and, following a single PCR, detect the results via a single universal tag DNA chip array. The application of MIP to generate a haplotype map of Chromosome 12 in CEU families (The International HapMap Consortium 2003) as part of the Human HapMap project has yielded 3,509,052 genotypes from 38,429 assays. In this study we analyze the important features of the assay performance that will enable MIP to be effectively used for many future largescale studies.

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.

Introduction: Our group has been developing methods to quantitatively detect mutant BRAF DNA in the plasma of metastatic melanoma patients, with the aim of developing new biomarker assays. In this study we evaluated the potential of a probe-based, droplet digital PCR (ddPCR) assay to quantitatively measure copies of DNA encoding the mutant BRAFV600E and wild-type alleles in the plasma of patients undergoing treatment with a BRAF inhibitor. We previously showed this assay to be 100% specific with a sensitivity to detect 1 mutant copy among 10,000 wild-type copies when sufficient input DNA was used. Methods: Ten patients with BRAF-mutant stage IV melanoma were prospectively studied. At least 3 serially-collected plasma samples were available for each patient, corresponding to pre-treatment, best treatment response, and progression of disease as evidenced by PET/CT scan. DNA was extracted from plasma samples and concentrations were measured using a Qubit fluorometer. The relevant BRAF region was assayed using 24 to a maximum of 337 ng of DNA from each sample, using multiple replicate wells for dilute samples to maximize the probability of detecting BRAF mutant copies. Specific Taqman 5′ hydrolysis probes for wild-type and mutant BRAF were employed along with common primers to measure the number of copies of each amplicon/reaction. The amount of amplifiable DNA was measured using RPP30 as a control gene. Results: All 10 patients had partial responses. We analyzed 35 plasma samples, of which 30 had a minimum of 4ml of plasma. Qubit-based DNA concentrations ranged from 6.9 to 1158 ng/ml. All samples yielded positive amplification of the BRAF and RPP30 amplicons. Total BRAF DNA concentrations ranged from 719 to 156,920 copies/ml plasma. Mutant BRAF DNA concentrations ranged from 0 to 13,028 copies/ml. The fraction of BRAFV600E amplicons ranged from 0 to 45%, with a minimal detected mutant fraction of 0.01%. In 8/10 patients, levels of mutant BRAF fell with clinical response and/or rose with disease progression. In 3 of these 8 patients, samples drawn 10 days to 2 months prior to imaging studies showed increased levels of BRAF mutant DNA prior to evidence of disease progression. In 1 of the remaining 2 patients levels of total BRAF DNA tracked with clinical outcome better than levels of mutant BRAF DNA. In 1/10 patients neither levels of mutant nor total BRAF DNA tracked with clinical responses. Conclusions: These results demonstrate that a ddPCR assay is able to quantitatively measure total and mutant BRAF DNA in the plasma of patients with stage IV melanoma. Levels of mutant and total BRAF DNA tracked with disease progression showing promise for this assay as a potential biomarker that may predict disease progression. Analyses of additional tumor-derived mutations in plasma DNA, and more precise assessments of disease response, are underway to determine optimal biomarkers to incorporate into melanoma clinical trial and patient care protocols. Citation Format: David Polsky, Jyothi Sakuntala Tadepalli, Gregory Chang, Nathaniel Fleming, Yongzhao Shao, Anna C. Pavlick, Iman Osman, Cindy Spittle, Manohar Furtado, Paula Stonemetz, Dawne Shelton, George Karlin-Neumann. Quantitative assessment of circulating BRAF DNA in stage IV melanoma patients undergoing BRAF inhibitor treatment. [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 2847. doi:10.1158/1538-7445.AM2014-2847

The recognition that tumors and other apparently homogeneous cell populations are often heterogeneous and display stochastic expression of their genes, has motivated the development of methods to profile gene expression in single cells. Among these, RNA-Seq is the most powerful for broad-scale profiling and discovery of genes and pathways influencing biological processes and states of interest. However, given the extent of sample manipulation required to prepare each RNA-Seq library – and the trade-off between sensitivity and sample throughput – it is important to validate discovery findings by orthogonal digital methods with comparable or greater sensitivity and accuracy. Droplet Digital PCR is such a method that can rapidly and cost-effectively replicate (or challenge) RNA-Seq results with minimal sample processing when defined targets are to be verified (Chen et al, 2012). Furthermore, ddPCR is performed in 96-well plates and is well-suited to high throughput studies of focused sets of genes in large numbers of single cells (e.g. hundreds in a day). Here we present a simple and robust workflow for profiling multiplexed, transcript targets in flow-sorted, Jurkat single-cells using Taqman 5’ probe hydrolysis assays. We demonstrate that Bio-Rad's QX100 & QX200 Droplet Digital PCR systems provide absolute counts of transcripts from >100,000 copies to More than 30 cell-cycle genes have been evaluated by ddPCR in exponentially growing Jurkat cells. Several genes, including CCNB1, CDK1 and DNM2, show an unusually broad range of transcript levels (over 2 logs), suggestive of their known phase-specific cell-cycle roles. Jurkat cells have been FACS sorted for either G0/G1 (“G1”) or G2/M (“G2”) single-cells using DyeCycle Orange to stain for DNA content, and are being assessed for phase-specific expression profiles. Preliminary results indicate that G2 single-cells are enriched for higher CCNB1 and CDK1 transcript levels as expected from their role in promoting early events in mitosis; however, even G2 cells still show expression levels spanning ∼2 logs, suggesting cell-cycle phase heterogeneity and/or bursty transcription within the sorted G2 population. While the average mRNA level of even the house-keeping gene, GAPDH, is several-fold higher in G2 cells as compared to G1 cells, transcripts for CCNB1 and CDK1 average ∼6-fold higher in G2 than in G1. In this work, we demonstrate both the analytical performance and the biological utility of droplet digital PCR for single-cell analysis. This same workflow could be used for fingerprinting and study of sub-populations in tumors. Citation Format: George Karlin-Neumann, Shenglong Wang, Camille Troup, Yann Jouvenot, Eli Hefner. Rapid and ultra-sensitive single-cell transcript profiling with droplet digital PCR (ddPCR): Application to cell cycle analysis. [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 3491. doi:10.1158/1538-7445.AM2014-3491

9019 Background: Management of melanoma suffers from a lack of robust biomarkers of disease activity. In this study, we tested the ability of droplet digital PCR (ddPCR) to quantitatively measure levels of circulating BRAF and NRAS mutant DNA in the plasma of metastatic melanoma patients undergoing treatment with BRAF-targeted therapy or immunotherapy. Methods: We analyzed plasma samples from 45 patients with stage IV melanoma prospectively enrolled in the NYU Melanoma Biorepository program. All patients were commercially genotyped for BRAF V600E. SNaPshot assays were used to identify NRAS Q61 mutations in BRAF-WT tumors. Each patient had at least 3 serially collected plasma samples including one drawn prior to treatment, one or more after treatment began and one upon signs of disease progression. ddPCR was used to measure mutant copies/ml of BRAF V600E and NRAS Q61K/L/R DNA in plasma samples. Results: Among 45 patients, 28 patients had BRAF and 8 patients had NRAS tumor mutations. We extracted DNA from 1...

The aim of this work was to develop a more sensitive and high throughput assay for measuring telomerase activity. Telomeres are the protective structures at the ends of chromosomes consisting of 6 bp repeat sequences. In young cells, these regions can be as long as 15kb and act as caps which protect the DNA ends. These ends naturally degrade with each passing cell division, usually losing 25-200 base pairs per division. Once they are shortened below a critical length (estimated to be 200-300 bp) the cells arrest and become senescent, or “old”. Telomeres can be thought of as a cellular or mitotic clock. Once the clock has wound down, the cells either die or pass through crisis and become immortal. One of the mechanisms of immortality is the activation of the enzyme, telomerase. Telomerase is the endogenous reverse transcriptase responsible for adding repeats to telomeres, rewinding the clock and enabling a cell to continuously divide. Abundant telomerase activity is found in fetal and adult stem cells, germ cells, and cancer. It is also present at much lower levels in non-stem cells, such as immune cells, but these levels can be difficult to measure reliably. The telomerase repeat amplification protocol (TRAP) measures the presence of active telomerase by measuring the activity of the enzyme on a starting DNA template, which is then amplified by PCR. For samples with abundant telomerase activity, SYBR qPCR assays provide high throughput. However, the current most sensitive method of detection still uses radioactivity, laborious PAGE sequencing gels, and densitometry to quantify telomerase. Here we explore using droplet digital™ PCR (ddPCR™) to provide absolute quantification of telomerase activity. This was achieved through single-molecule counting of telomerase-extended templates that were partitioned into droplets, amplified by PCR and detected by fluorescence droplet flow cytometry. Analysis of control samples suggests ddPCR is at least an order of magnitude more sensitive than TRAP radiography and is more amenable to higher throughput analysis. We extend throughput, sensitivity, and the range of biological samples that can be analyzed for telomerase activity. Citation Format: Dawne Shelton, Jack Regan, George Karlin-Neumann, Jue Lin, Elizabeth Blackburn. TRAPing telomerase activity using droplet digital PCR. [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 LB-253. doi:10.1158/1538-7445.AM2013-LB-253

Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency. 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 3399. doi:1538-7445.AM2012-3399

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples.

BackgroundA major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.ResultsUsing an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.ConclusionMIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.