Although the clinical demand for bioengineered blood vessels continues to rise, current options for vascular conduits remain limited. The synergistic combination of emerging advances in tissue fabrication and stem cell engineering promises new strategies for engineering autologous blood vessels that recapitulate not only the mechanical properties of native vessels but also their biological function. Here we explore recent bioengineering advances in creating functional blood macro and microvessels, particularly featuring stem cells as a seed source. We also highlight progress in integrating engineered vascular tissues with the host after implantation as well as the exciting pre-clinical and clinical applications of this technology.
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.
In the originally published edition of our Forum, a draft version of Figure 1 appeared by mistake. The correct, final version of Figure 1, which differs from the original only in terms of panel layout, has since been replaced in the article online. We apologize for any confusion this may cause.
The caption for Figure 5 reads: One-color SNV quantification. (A) Primers are designed with the single nucleotide variant at the 3′ end of the complementary region. Noncomplementary tails of varying lengths are then added to the 5′ end and amplified with a universal reverse primer. (B) 1:4 mixture of MUT/WT BRAF template amplified with mutant primers with the short tail and wild-type primers with the long tail. (C) Swap: 1:4 mixture of MUT/WT BRAF template amplified with wild-type primers with the short tail and mutant primers with the long tail. (D) Serial dilution of mutant BRAF template (LS411N) into wild-type (Human male control). Theoretical % mutant was calculated from TaqMan measured concentrations of mutant and wild-type template. The assay was performed with the EvaGreen primer mix from (B). (E) The red border regions provide a magnified view of three data points on the lower end of the dilution series from (D).However, the caption for Figure 5 should read: One-color SNV quantification. (A) and (B) Primers are designed with the single nucleotide variant at the 3′ end of the complementary region. Noncomplementary tails of varying lengths are then added to the 5′ end and amplified with a universal reverse primer. (C) 1:4 mixture of MUT/WT BRAF template amplified with mutant primers with the short tail and wildtype primers with the long tail. (D) Swap: 1:4 mixture of MUT/WT BRAF template amplified with wild-type primers with the short tail and mutant primers with the long tail. (E) Serial dilution of mutant BRAF template (LS411N) into wildtype (Human male control). Theoretical % mutant was calculated from TaqMan measured concentrations of mutant and wild-type template. The assay was performed with the EvaGreen primer mix from (C). (F) The red border regions provide a magnified view of three data points on the lower end of the dilution series from (E).
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
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
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