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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.

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.

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 …

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.

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.

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

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.

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.