Mutated DNA Fragments Research Articles

Electrochemiluminescent CdS Quantum Dots Biosensor for Cancer Mutation Detection at Different Positions on Linear DNA Analytes.

PCR-based techniques routinely employed for the detection of mutated linear DNA molecules, including circulating tumor DNA (ctDNA), require large nucleotide sections on both sides of the mutation for primer annealing. This means that DNA fragments with a mutation positioned closer to the extremities are unlikely to be detected. Thus, sensors capable of recognizing linear DNA with characteristic mutations closer to the ends would be advantageous over the state-of-the-art approaches. Here, an electrochemiluminescence-resonance energy transfer (ECL-RET) biosensor comprising capped CdS quantum dots and hairpin DNA probes labeled with Au nanoparticles was developed for the detection of epidermal growth factor receptor (EGFR) ctDNA carrying the critical T790M lung cancer mutation. The ECL-RET system detected different DNA molecules including single-stranded 18-nucleotides (nt) and 40-nt as well as double-stranded 100-nt with the single nucleotide polymorphism (SNP) coding for T790M located either in the middle or only 7 nt from one end. For all target DNA, the sensor's limits of detection (LODs) were in the aM range, with excellent selectivity. It was the case of 100-nt target linear ctDNA fragments with LODs of 8.1 and 3.4 aM when the EGFR T790M SNP was either in the middle or at the end, respectively. These results show that ECL-RET systems can sense mutations in DNA fragments that would remain undetected by standard techniques.

Single-cell RNA sequencing reveals the cellular and molecular characteristics of high-grade and metastatic bladder cancer.

Metastatic bladder cancer (BC) has the highest somatic mutation frequency and recurrence rate of all tumors. However, the cellular and molecular characteristics of BC remain unclear. We performed single-cell RNA sequencing (scRNA-seq) on the samples of paracancerous normal tissue (PNT), primary tumor (PT) and lymph node metastasis (LNM). The proportions and gene expression profiles of different cell types in the tumor microenvironment (TME) were investigated. In total, 50,158 cells were classified into six populations. Malignant cells of PT and LNM exhibited large mutant DNA fragments, while the cell phenotypes and gene expression profiles differed during differentiation. Metastasis was associated with a poorer prognosis than PT. Tumor-associated stromal cells and inhibitory immune cells were the main cell populations in PT and LNM. Cell-cell communication analysis revealed the roles of signaling pathways of inflammatory cancer-associated fibroblast (iCAF) and tumor-associated macrophage (TAM) in exhaustion of T cells. In addition, iCAF may recruit TAM to promote formation of the TME earlier than the differentiation of tumor cells. This study through scRNA-seq enhanced our understanding of new features about the cellular and molecular similarities and differences of high-grade and metastatic bladder cancer, which might provide potential therapeutic targets in future treatment.

Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries—including Germany—assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.

Molecular Approaches for Detection of Rifampin-Resistant Mycobacterium tuberculosis and Its Associated Risk Factors among General Population

GeneXpert MTB/RIF is a molecular technique for the diagnosis of tuberculosis. This method not only detects M. tuberculosis but also identifies the absence or presence of different drug resistant mutations in DNA fragments like rifampicin resistance. In this study, about 200 septum samples were collected from tuberculosis suspected people of Isfandyar Bukhari District Head Quarter Hospital Attock for screening and determination of Mycobacterium tuberculosis and multi-drug-resistant tuberculosis. The samples were screened for the presence of M. tuberculosis using Fluorescence Microscopy staining followed by GeneXpert MTB/RIF assay. The results were further confirmed by Line Probe Assay and Mycobacterium Growth Indicator Tube 960 techniques. In a total of 200 samples, 45(22.5%) were found positive, 155 (77.5%) were classified negative on culture while 38(19%) were positive and 162(81%) were negative on FM, 33(16.5%) were positive and 167(83.5%) were negative on ZN staining microscopy and 47(23.5%) were noted positive and 153 (76.5%) were negative on GeneXpert. The overall sensitivity of GeneXpert was 07% higher than that of smear microscopy and 4.2 % higher than culture.Different risk factors reported for TB are; malnutrition (23.40%), health care workers (14.89%), immunosuppression (12.76%), unimmunized with Bacille Calmette-Guérin (BCG) vaccine and contact with infected persons (10.63%), diabetes mellitus (8.51%), poverty and foreign-born children are (4.2%). It is concluded that the most common risk factor for TB was malnutrition (23.40%) and the foreign-born children (4.2%) were least affected. GeneXpert assay for identification of tuberculosis was found more sensitive than fluorescence microscopy and for isolation of MTB and rifampicin resistance, GeneXpert RIF assay can efficiently be usedwhich is simple, robust and efficient with minimum handling technique.

DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.

PCR đặc hiệu allele tích hợp công nghệ amplification refractory mutation system (Dsa-Pcr-Arms) phát hiện đột biến jak2 v617f và Carl trên bệnh nhân có hội chứng tăng sản tủy

BIDIRECTIONAL ALLELE SPECIFIC PCR INTEGRATED WITH AMPLIFICATION REFRACTORY MUTATION SYSTEM –(DSA-PCR-ARMS) FOR ANALYZING CARL AND JAK2 V617F MUTATIONS IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASM Bachground: Jak2 V617F and CARLmutation arerecognized as essential molecular genetic markers in diagnostic definition of myeloid proliferative neoplasm (MPN). Objective: to establish an income relevant Allele-specific-PCR-ARMS (DSA-PCR-ARMS) assays to identify Jak2 V617F, CARL mutations in MPN patients. Subjects: 64 MPN patients’ samples, one V617F positive referrance samples and one CALR mutant positive sample. Methods: dilution series of 100%, 50%, 25%, 5%, 0,5% and 0% mutant alleles were created as pseudo-samples establish the detection limits and the technical specificity of DSA-PCR-ARMS assays in identifying the mentioned genetics markers. Result: Our approach can detect presence of 0.5 % V617F mutant DNA fragment; beside that just by one gel-based PCR reaction, we can differentiate the deletion/insertion CARL carriers from the wild type counterpart. By applying our approachesto a study cohort of 50 MPN cases, a sensitivity of 64% and specificity of 100% was recorded for our Jak2 V617F detection assay and we also detected 3 CARL mutation case amongst the 10 V617F negative cases. Conclusion: The in-house of Jak2 V617F/CARL mutation detection protocols have been successfully established in our hospital. Key words: Jak2 V617F, CARL, myeloid proliferative neoplasm (MPN).

Molecular detection of H. pylori antibiotic‐resistant genes and molecular docking analysis

To explore the mutation characteristics of H. pylori resistance-related genes to antibiotics of clarithromycin (CAM), levofloxacin (LVX) and metronidazole, 23SrRNA, gyrA, gyrB, rdxA, and frxA genes were amplified and sequenced, respectively. Molecular docking study was performed to explore molecular interactions between chemotherapeutic agents and target proteins. In the CAM-resistant strains, the mutation rate in site A2143G was 74.2% (n=23). The interactions in sites of G1949A, C1953T, and G2211T with CAM were weaker after mutation. In the LVX-resistant strains, the mutation rates in 87 (N to K/I) and 91 (D to N/Y/G) of gyrA were 28.6% (n=16) and 12.5% (n=7), respectively. We could infer by docking studies that N87 K/I, D91Y/G/N, D99N, and D143E mutations in GyrA protein all had weakened interaction with LVX. The mutation types of RdxA protein consisted of protein truncation caused by premature stop codons (n=26, 33.3%), frameshift mutations (n=8, 10.3%), FMN-binding sites (n=16, 20.5%), and the others (n=11, 14.1%). Docking analysis showed that some mutations in those four types of RdxA protein could reduce interaction with MNZ, and play a significant role in antibiotic resistance. What's more, we performed natural transformation with some of these mutated DNA fragments to see if they really impact susceptibility to antibiotics. Our study suggested that in addition to the reported mutation sites, H. pylori antibiotic resistance in this region may also be associated with changes in some new sites. Our study provides novel ideas and methods for the identification of H. pylori resistance-related mutations, and also clues and basis for further investigation on specific molecular mechanism.

Abstract 1378: Digital PCR probe library for TP53 mutations in early relapse prediction

Abstract Precise relapse prediction in cancer patients is urgently needed as conventional serum tumor markers do not predict relapse in a timely manner. Circulating tumor DNA (ctDNA) is a new class of quantitative biomarkers that reflect tumor burden. The ctDNA is usually found as mutated DNA fragments in the blood, which are generally consistent with the mutations found in the concomitant tumor in the body. Variant allele frequency (VAF) of ctDNA is usually very low and ranges from 0.001 to 1% even in advanced cases. To quantify absolute copy number of ctDNA at the very low range, digital PCR (dPCR) is one of the most practical methods. However, specific primers/probes for dPCR must be designed individually as tumor mutation varies between individual patients. The TP53 gene is the most frequently mutated in human cancer and 90% of the mutations occur in its DNA binding domain coding region. According to the COSMIC database, there are 1,284 unique mutations from a total of 25,376 recorded mutations in the DNA binding domain. The majority (77%) of the 1,284 unique mutations are not recurrent and thus 42 unique mutations cover 50%, 132 unique mutations cover 70%, and 426 unique mutations cover 90% of the total mutations. Here we have designed a dPCR probe library for the set of 132 unique mutations to cover 70% of TP53 mutations in the DNA binding domain using Hypercool Primer & ProbeTM technology. With this technology, modified bases, 2-Amino-dA and 5-Methyl-dC, were substituted in adenine and cytosine positions, respectively. This modification increases the Tm, thus allowing for highly specific amplification despite short PCR products of 70-90 bp. The dPCR library allows "off-the-shelf", highly sensitive ctDNA tracking once the TP53 mutation is identified in the target tumor (e.g., surgical specimens). In our pilot cases, we found TP53 mutations from 97% (34/35) of esophagus, 70% (7/10) of stomach, and 93% (13/14) of colorectal primary tumors using next generation sequencer (NGS). There were 40 unique mutations for which all mutations were covered with our TP53 dPCR probe library. We then validated the dPCR library for ctDNA in terms of: (a) the ability to detect the target mutation; (b) tumor genomic heterogeneity; and (c) early detection of relapse. Of >500 DNA samples from primary cancers and patient plasma, 98% of probes tested were confirmed to detect the target mutation properly. Multiregion sequencing of stomach and colon cancers by NGS revealed that 85% (17/20) of tumors exhibited TP53 founder mutations, offering a high chance that the corresponding ctDNA was detected in plasma. Among cases who experienced relapse confirmed by CT scan, ctDNA elevation was seen approximately 6 month prior to the confirmation by CT scan. Overall, the sensitivity of VAF using dPCR with the probes was as low as 0.001%. These results suggest that the TP53 dPCR probe library can potentially be used for a wide range of human cancers as a new class of biomarkers for early relapse prediction. Citation Format: Satoshi S. Nishizuka, Mizunori Yaegashi, Noriyuki Sasaki, Takeshi Iwaya. Digital PCR probe library for TP53 mutations in early relapse prediction [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 1378.

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products.

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection.

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.

Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.

IDENTIFICATION OF ANTIRETROVIRAL MUTATION IN PROTEASE AND REVERSE TRANCRIPTASE INHIBITOR IN HUMAN IMMUNODEFICIENCY VIRUS-1 OF HIV/AIDS PATIENTS IN MIMIKA REGENCY, PAPUA

Treatment with RT Inhibitors has been used as first line program in Mimika. Regular use of antiretroviral drugs can lower the amount of the virus, but after that HIV can become resistant to the drugs given. Viral resistance to the drugs can occur because of a mutation. If the resistance-associated mutations occur in the DNA of the virus, then the ARV provided will no longer be effective. The aim of this study was to identify the presence of resistance-associated mutations in DNA fragment that encodes the protease and reverse transcriptase. This study used cross sectional design with 84 subjects who had received antiretroviral for > 6 months. The study was conducted in Mitra Masyarakat Mimika Hospital. Laboratory process included extraction, RT-PCR, electrophoresis and sequencing. Data analysis used resistance interpretation algorithms in HIV Database. Results showed that 1 subject who did not receive protease therapy was identified as having minor mutation L10V, 1 subjects receiving NRTI inhibitors had mutation M184V motive and 1 subjects with M41L motive and 1 subjects who received NNRTI inhibitor therapy identified as having mutated Y181C and V108I motive. In conclusion, mutant HIV-1 related to ARV resistance has been identified in two subjects who had received antiretroviral therapy in Mitra Masyarakat Mimika Hospital.

THE EXISTENCE OF GENE DNA POLYMERASE MUTATION FROM POSITIVE HEPATITIS B SAMPLES IN BANDUNG, INDONESIA

Objective: To complete mutation existence in reverse transcriptase domain of the viral Polymerase.Methods: The study was done by amplification step of viral polymerase gene fragment, agarose gel electrophoresis, PCR product purification with GFX column kit, sequencing and sequencing result analysis. The samples were derived from Clinical Laboratory in Bandung Indonesia.Results: The result showed mutations in DNA fragment encoding for RT domain viral polymerase in sample 6, 7 and 8. There were mutations leading to amino acid substitution L526S in sample 6, D551E in sample 7 and D552E in sample 8.Conclusion: D551E and D552E substitution occurred in YMDD motif RT DNA polymerase that produced YMDE mutant. L526S, D551E and D552E were estimated as antivirus-resistance mutants that have never been reported before.

Feasibility of multiplexed gene mutation detection in plasma samples of colorectal cancer patients by mass spectrometric genotyping.

Mutation analysis of circulating tumor DNA (ctDNA) has recently been introduced as a noninvasive tumor monitoring method. In this study, we tested the mass spectrometric-based MassARRAY platform for multiplexed gene mutation analysis of plasma samples from colorectal cancer (CRC) patients. A total of 160 patients, who underwent curative resection of either primary or metastatic CRC harboring KRAS mutations between 2005 and 2012, were included. Circulating DNA was isolated from plasma was analyzed on the MassARRAY platform with or without selective amplification of mutant DNA fragments. Tumor-specific KRAS mutations were detected in 39.6% (42/106) of patients with distant metastasis, and in 5.6% (3/54) of patients without distant metastasis. Selective amplification of the mutant allele increased sensitivity to 58.5% (62/106) for patients with distant metastasis, and 16.7% (9/54) for patients without distant metastasis. These mutation detection rates were no less than those of droplet digital polymerase chain reaction. Among patients with distant metastasis, detectable plasma KRAS mutations correlated with larger primary tumors and shorter overall survival rate (P = 0.014 and P = 0.003, respectively). In addition, activating PIK3CA mutations were detected together with KRAS mutations in two plasma samples. Taken together, massARRAY platform is a cost-effective, multigene mutation profiling technique for ctDNA with reasonable sensitivity and specificity.

The reliable assurance of detecting somatic mutations in cancer-related genes by next-generation sequencing: the results of external quality assessment in China.

To evaluate the proficiencies of laboratories utilizing next-generation sequencing (NGS) to detect somatic mutations in cancer-related genes, an external quality assessment (EQA) was implemented by the National Center for Clinical Laboratories of China in 2015. We prepared a panel of samples that comprised eight samples made by mixing synthetic mutated DNA fragments with normal human genomic DNA and one reference sample containing only genomic DNA. We validated our sample panel, and then distributed it to laboratories across China. We received complete results from 64 laboratories. The performances of 51.6 % (33/64) respondent labs were acceptable and 26.6 % (17/64) of the labs returned perfect results. In total, 449 mistakes were reported, including 201 false-negatives (201/449, 44.8 %) and 222 false-positives (222/449, 49.4 %) and 26 slightly discordant results (26/449, 5.8 %). We believe these unsatisfactory results and varied performances are mainly due to the enrichment methods used, the diverse sequencing chemistries of the different NGS platforms, and other errors within the sequencing process. The results indicate that our sample panel is suitable for use in EQA studies, and that further laboratory training in targeted NGS testing is urgently required. To address this, we propose a targeted NGS workflow with details on quality assurance procedures according to the current guidelines.

Research of methods to detect genomic mutations induced by CRISPR/Cas systems

The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.

The dynamic range of circulating tumor DNA in metastatic breast cancer.

IntroductionThe management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer.MethodsIn this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments.ResultsAlthough all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer.ConclusionsThe dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0421-y) contains supplementary material, which is available to authorized users.

A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.

Plasma cell-free DNA and fraction of circulating KRAS mutations as prognostic in patients with metastatic colorectal cancer.

490 Background: It has been suggested that detection of circulating tumor DNA may be relevant in patients with metastatic colorectal cancer (mCRC). The main objective of the present study was to evaluate a method based on the TaqMan Mutation Detection Assay (TMDA) for the detection of circulating KRAS mutations in mCRC patients. Moreover, we also investigated the prognostic impact of the plasma cell-free DNA and the fraction of circulating KRAS mutations. Methods: The study was conducted from April to July 2013 and plasma samples were prospectively collected in a series of 35 mCRC patients treated with chemotherapy (CT). QIAamp Circulating Nucleic Acid kit was used for DNA extraction and Quant-iT High Sensitivity dsDNA Assay for cf-DNA quantification. Detection of circulating tumor DNA was based on the KRAS mutations detected in tumour and was performed in plasma by the castPCR Technology TMDA. Response to CT was assessed according to RECIST criteria. The results of plasma cf-DNA and level of mutant DNA fragments were correlated with response and 3-months survival. Results: We isolated and quantified plasma cf-DNA in all patients with a mean concentration of 106 ng/mL. Among them, 18 were wild-type and 17 mutated for KRAS in the tumour. Detection of circulating KRAS mutations was performed with TMDA in 23 patients (10 KRAS wild-type and 13 KRAS mutated). The sensitivity was 62% (8/13) and specificity 100% (0/10) with a level of circulating mutant DNA fragments ranging from 0 to 29%. Plasma cf-DNA and level of circulating mutant DNA were both significantly correlated with the 3-months survival (mean 36 versus 524 ng/mL, p=0.0015 and 2% versus 29%, p<0.0001). There was a non significant trend for response to CT (respectively p=0.14 and p=0.12). Conclusions: TMDA method is a simple, accurate and non-invasive tool for the detection of circulating tumor DNA. Our preliminary results also suggest that plasma cf-DNA and fraction of mutant DNA fragments could be prognostic markers in mCRC patients.