Laura Miotke

High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR

In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

BackgroundGastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.ResultsBoth the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.ConclusionsWe document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffin embedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas.

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy.

Background Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far. Methods and Results Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit. Conclusion Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.

Abstract 1380: Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring

We have developed a new molecular assay that utilizes digital PCR to detect and quantify cancer mutations within poor quality and limited quantity samples such as archival tissue DNA and circulating tumor DNA (ctDNA). This digital PCR assay is highly sensitive and is capable of detecting the targeted mutant fraction at an individual DNA molecule resolution. Thus, this assay is ideal for applications where DNA is in low abundance. An example is where DNA is shed from tumors that can be extracted from the plasma fraction of routine blood draws. The resulting circulating DNA (ctDNA) is extremely low in concentration, which makes it a prime candidate for our highly sensitive molecular assay. Our assay incorporates small amplicon PCR primers and can be configured for nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. Mutation quantitation relies on two DNA primer sets that are identical with the exception of the mutant or wild type specific base at the 3’ end of the “detecting” primer sets, to amplify the genomic region of interest. Through the addition of artificial 5’ non-complementary tails to our mutant and wild type specific “detection” primers, we are able to consistently differentiate between droplets that contain the mutant or wild type alleles based upon their differential amplicon lengths. The synthetic amplicon extension tails minimize bias within the PCR reaction by allowing our primers to target an identical region of genomic DNA, with the exception of the single nucleotide variant specific base at the 3’ end of the detecting primer. The dPCR technology allows the standard PCR reaction to be partitioned into 20,000 independent wells; we can then assay each individual droplet to assess whether the individual DNA molecule partitioned into the droplet is “wild-type” or the target “mutant”. The resulting data provides an absolute count of mutant and wild-type templates in a given patient sample. We have optimized this new molecular assay technology for quantitatively measuring clinically actionable mutations including BRAF V600E, KRAS G12D among others. We have successfully validated the sensitivity, specificity and reproducibility of our assays through controlled cell line DNA mixed dilution samples ranging from 66% mutant to 0.1% mutant in each of our optimized primer sets. Our assay improves the limit of detection to seven DNA molecules containing a mutation among a total number of 7,000 genome equivalents. Because our assay is sensitive down to the single DNA molecule resolution, we have also been able to reduce the amount of clinical sample DNA required to determine the presence of clinically actionable mutations such as those occurring in KRAS or BRAF. We are testing this extremely low cost, highly sensitive diagnostic technology for detecting nearly any cancer mutation from longitudinal samples of ctDNA. Citation Format: Christina M. Wood, Billy Lau, Laura Miotke, Hanlee P. Ji, Stephanie Greer. Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring. [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 1380.

Abstract 1507: Highly sensitive and specific digital quantification of cancer genetic aberrations

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Genetic aberrations play a major role in tumor development and progression. As these clinically relevant genetic aberrations are discovered, it becomes of pressing urgency to efficiently, rapidly and sensitively detect these targets in individual patient tumor samples. Droplet digital PCR (ddPCR) is a robust technology for quantitating genetic variation with high sensitivity from minimal amounts of nucleic acid. Compared to the highly variable quantitative real-time PCR (qRT-PCR), ddPCR eliminates relative standards and has the advantage of measuring mutant and wild-type targets within the same well. Commonly, this is achieved with the use of a two-color fluorescent oligonucleotide probe (TaqMan) design, where the mutant is represented by a FAM probe and the wild type by a VIC or HEX probe. However, this approach is cumbersome and requires a significant amount of optimization. In this study we present an alternate quantification method for assessing CNVs and SNVs using a non-specific DNA binding dye. Instead of two colored probes, our design manipulates the length of the region of interest (ROI) and control amplicons to distinguish between their fluorescent signals. The dye binds in greater amount to the longer length target giving a higher fluorescent signal than the mutant target and both populations are easily quantifiable. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay. The ability to customize this assay for a variety of functions is a major advantage of using a single-color dye and thus we can create highly sensitive assays for any gene. Here, we demonstrated the effectiveness of this method by assessing copy number of the proto-oncogene FLT3, a target for the small molecule inhibitor Sunitinib. We established accurate quantitation of a FLT3 copy number change in a tumor sample harboring a 1.5-fold amplification diluted to 20% in a normal sample. Additionally we explored the common V600E point mutation in BRAF and an activating mutation in HER2, a potential therapeutic target for cancers lacking HER2 amplification. Our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample and in the case of HER2, distinguish between wild-type and two different mutant alleles. Aside from single base pair mutations and copy number analysis we used this assay to detect single base pair insertions and deletions. Finally, due to the sensitivity of ddPCR, the applications of this technique include the targeted analysis of small biopsies and circulating nucleic acid. We are also determining the improvement in performance with the incorporation of locked nucleic acids (LNA) in increasing specificity and sensitivity in lower quality samples. Citation Format: Laura K. Miotke, Billy Lau, Rowza Rumma, Hanlee Ji. Highly sensitive and specific digital quantification of cancer genetic aberrations. [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 1507. doi:10.1158/1538-7445.AM2014-1507

Abstract 3584: Analysis of colorectal intratumoral genetic heterogeneity by high efficiency and rapid deep targeted sequencing

Metastasis is the most frequent cause of morbidity and mortality in cancer. Metastatic disease lethality is often the result of cancer progression despite treatment. Therapeutic resistance is seen nearly in all cases of metastatic cancer. Genetic studies relying on deep sequencing have shown that many cancers are genetically heterogeneous as a result of diverse clonal populations with delineating and unique set of mutations. Several studies suggest that resistance mutations are present in tumors before the start of treatment and via evolution, clonal populations expand under therapeutic selection. However, these minor allelic mutations that define clonal populations are not typically detectable with typical sequencing coverage (20-50X) employed in whole genome approaches. Relying on an innovative programmable targeting method that enables us to rapidly configure nearly any region of the human genome and efficiently sequence cancer genomes, we are determining the extent of intratumoral genetic heterogeneity and clonal diversity in colorectal cancer. Our study involves over 120 patients with available clinical data and a subset of matched tumors indicative of tumor progression (e.g. tumor-normal, premalignant-malignant, malignant-metastasis). Via an integrative analysis of TCGA, COSMIC and other genomic data sets of colorectal cancer, we identified the top ranking 53 cancer genes prone to mutation and associated with advanced colorectal cancer by. These top genes are found in known cancer pathways such as the WNT and RAS/RAF pathway and we are analyzing the exons of these genes for mutations, insertions and deletions with a sequencing depth of at least 1000x. Preliminary results have shown that we can readily reach sequencing depths of 7000x with our automated targeting approach and that a minimal sequencing depth of 1000x will enable detection of aberrations present in the sample below 1%. Samples are being analyzed for the overall level of heterogeneity and correlation of clonal mutations to clinical outcome. For optimal personalized treatment of cancer patients, the analysis of intratumoral genetic heterogeneity may be useful in predicting treatment response and metastatic potential of any given primary colorectal cancer. Citation Format: Erik S. Hopmans, Hojoon Lee, Laura Miotke, Rowza Tur Rumma, Sue Grimes, John M. Bell, Hanlee P. Ji. Analysis of colorectal intratumoral genetic heterogeneity by high efficiency and rapid deep targeted sequencing. [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 3584. doi:10.1158/1538-7445.AM2014-3584

Abstract 3488: Surveying colorectal cancer genome for clinically actionable genomic amplifications with droplet digital PCR.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC New cancer therapies are increasingly geared towards exploiting critical genetic and genomic features specific to the tumor. These mutations and genomic aberrations are the basis for precision cancer medicine. Thus, rapid molecular characterization of clinical cancer samples has become increasingly important for cancer targeted therapy development. Our study addresses this need by identifying ‘druggable’ gene amplifications in colorectal cancer (CRC). Along these lines, we developed an accurate and rapid droplet-digital PCR (ddPCR) assay to analyze cancer-specific copy number variations (CNV) in 13 genes with oncogenic function that are the targets of specific cancer therapies. These genes were selected based on the availability of approved and early-phase targeted therapeutics that inhibit their oncogenic function. Using TCGA data, we vetted our targets by identifying frequent CRC amplifications and a correlation with gene expression. The ddPCR method involves emulsifying matched-normal cancer sample DNA that provides specific advantages for highly sensitive and specific detection of cancer CNVs. Post-amplification, emulsion droplets are streamed single-file into a capillary that leads past a two-color detector, where the positive droplets for the target and reference genes are “counted” for quantitation. This technology requires nanogram amounts of genomic DNA, thus facilitating the study of clinical cancer samples from small biopsies. It works well with genomic DNA extracted from formalin fixed paraffin embedded tumor samples. Furthermore, we demonstrated high sensitivity for detecting copy number amplifications even in samples containing a prominent fraction of normal tissue. We are extending our analysis to the full 13 “druggable” gene targets and evaluating patterns of mutual exclusivity and co-occurrence among a cohort of over 200 CRC tumors with information on clinical outcome. For example, we have demonstrated FGFR1 amplifications in 5.2% of our CRC tumor population. For ERBB2 variations in the same cohort, we detected amplifications in 3.6% of our population. ERBB2 and FGFR1 amplification events displayed a mutually exclusive pattern of segregation (p value <0.0001 per Chi-squared test with Yates correction). This efficient and inexpensive assay offers a significant potential to extend personalized therapeutic options available to CRC patients. Citation Format: Rowza T. Rumma, Laura Miotke, Lincoln Nadauld, Georges Natsoulis, Michael DiMaio, Moe Jalali, Hanlee Ji. Surveying colorectal cancer genome for clinically actionable genomic amplifications with 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 3488. doi:10.1158/1538-7445.AM2013-3488

Abstract 4130: Detection of FGFR1 and FGFR2 amplification in triple-negative breast cancer using digital droplet PCR and DNA-based microarrays.

Background: Triple-negative breast cancers (TNBC) lack targeted therapeutic strategies and identification of potential oncogenic targets is imperative. Fibroblast growth factor (FGF) pathway has been implicated in mammary tumorigenesis and is a potential target in TNBC. Amplification of FGFR2 (fibroblast growth factor receptor 2), identified using genomic studies, has been reported in 4% of TNBCs. Selective FGFR inhibitors are in clinical development and patient selection for these trials is important. Preclinical data suggests that cell lines with FGFR amplification are sensitive to FGFR inhibitors. The aim of this study was to identify FGFR1 and FGFR2 amplification in TNBCs using a quantitative and sensitive methodology of digital droplet polymerase chain reaction (ddPCR) and compare to our results from a DNA-based microarray analysis. Methods: Fresh-frozen breast tumor core biopsies were collected from patients enrolled onto a TNBC neoadjuvant clinical trial. DNA from each tumor and matched germline-derived sample (n=56 pairs) was hybridized to Affymetrix Molecular Inversion Probe (MIP) array to determine copy number variation (CNV). ddPCR was used to assess amplification in FGFR1 and FGFR2 in 11 and 53 tumor/ germline DNA sample pairs respectively. The ddPCR technology utilizes TaqMan chemistry PCR primers and probes specific for FGFR1 and FGFR2. It quantitates copy number by streaming emulsion droplets single-file into a capillary that leads past a two-color detector, where the positive droplets for the target and reference genes are quantified. Copy numbers for target genes are calculated by comparing to an internal control (ultra-conserved region of chromosome 1). Results: CNV in FGFR1 and FGFR2 was assessed in 56 TNBCs. No FGFR1 amplifications were identified in any of the samples. FGFR2 amplifications were identified in 2/56 (4%) tumor samples. ddPCR was used to assess quantitative copy number in 53 paired tumor/ germline DNA samples for FGFR2 and 11 paired samples for FGFR1. High amplifications with 6-8 copies of FGFR2 were identified in 2/53 (4%) of the TNBCs. These two samples were the same as the ones identified to have a high copy gain by CNV analysis. No FGFR1 amplifications were identified by ddPCR and this was consistent with our CNV analysis result. Conclusions: Our FGFR amplification results were in congruence using two different methodologies. No FGFR1 amplification was identified in the TNBC samples assessed and FGFR2 amplification was identified in 4%. ddPCR was done on fresh-frozen TNBCs in this study but this technology can be applied to formalin fixed paraffin embedded tumors as well. ddPCR can detect multiple cancer genome amplifications and has a potential for large scale application. There are several FGFR inhibitors in clinical trials and ddPCR methodology is a clinically applicable strategy for identifying patients with FGFR amplification. Citation Format: Shaveta Vinayak, Lincoln D. Nadauld, Laura Miotke, Rowza T. Rumma, Melinda L. Telli, Hanlee P. Ji, James M. Ford. Detection of FGFR1 and FGFR2 amplification in triple-negative breast cancer using digital droplet PCR and DNA-based microarrays. [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 4130. doi:10.1158/1538-7445.AM2013-4130