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|Cheap dissertation chapter proofreading sites uk||Genes Chromosomes Cancer 58— J Clin Endocrinol Metab —9. The main aim was to obtain preliminary results regarding the effects of biomarkers on the different clinical variables included in the study, as well as to interrogate the possible implications of the tumour bulk, as tumour volume and number of metastatic sites, with the biomarkers or the clinical parameters included, as response or survival. Next-generation sequencing-based detection of circulating tumour DNA after allogeneic stem cell transplantation for lymphoma. Publish with us Thesis+anti+synthesis authors Thesis+anti+synthesis manuscript. All clinical validations should be included in the outlined validation protocol, including the entire range of clinical samples expected for the patient population, and, if appropriate, validation of the bioinformatics pipelines.|
He was able to separate the nucleic acids using a technique called adsorption chromatography. In addition, Chargaff also pointed out that in any section of DNA, the number of A residues was always equal the number of T residues and that the number of C residues were always equal to the number of G residues. Later, Watson and Crick would correctly propose that A and T actually pair together and that G and C pair together due to hydrogen bonding , which is known as Watson-Crick base pairing.
By analyzing the chemical structures of these molecules, Watson and Crick pointed out that A and T both have two hydrogen bonds available, which is why they pair together. C and G have three hydrogen bonds available, which is the reason they pair together. Shortly after Chargaff was making his discovery, another significant discovery was being made by scientists Maurice Wilkins — and Rosalind Franklin — Their research illustrated that the DNA molecule had a helical shape and was made up of two strands that were connected by ladderlike rungs.
They were able to prove this by studying crystallized X-ray patterns of DNA. Therefore, Watson and Crick established that the molecular structure of DNA was in fact a double helix. This was significant because they were then able to explore and propose a model to explain how DNA worked. It should be pointed out that the structure of DNA was discovered based on the research and results of many scientists. Watson and Crick definitely made this significant discovery, but they gained much insight from the works of Chargaff, Wilkins, and Franklin.
A chromosome is a long, single piece of DNA that contains several genes; in some species 10 to 40 genes and in other species thousands or more genes can be present in just one chromosome. In eukaryotes, the chromosomes are organized structures that consist of DNA and special structural proteins called histones that wind, coil, and compact large DNA sequences so that they fit efficiently in the nucleus. The chromosome does not always stay condensed, but periodically relaxes and uncoils for replication and for the transcription of proteins.
In prokaryotic cells, the DNA is either organized in clusters with no nucleus or into small circular DNA molecules called plasmids, which do not contain histones. In viral genomes, very simple chromosomes are found and can be made out of DNA or RNA, which are short, linear or circular chromosomes that usually lack structural proteins.
In all animals, DNA in the chromosomes is packed by histones into globular aggregates known as a nucleosomes. In addition, it is now known that the genes that code for histones have no introns. The solenoids are then condensed into a chromatin fiber, which has histone H1 holding the core together. The chromatin fiber then folds into a series of loops that are held together by a central scaffold made of nonhistone chromosomal protein , and this configuration is called a looped fiber.
The looped fiber is then coiled to form the fully condensed heterochromatin of the chromosomes. The coiled and condensed heterochromatin pairs up with an identical copy of itself, and each of the two are referred to as a chromatid. Two identical chromatids are attached to each other by a centromere.
The centromere divides both chromatids into a long arm and a short arm. During cell division, microtubules attach to the centromere and align the chromosomes in the center of the dividing cell. The chromosomes are then split, yielding two identical cells—each with its own set of chromosomes.
All four arms of the chromosome two long arms and two short arms have a specialized cap known as a telomere, which has several functions e. The chromosomes also show a distribution of two types of bands. As mentioned earlier, the DNA molecule is composed of two purines A and G and two pyrimidines T and C , and all four are nitrogenous bases.
These nitrogenous bases are attached to a deoxyribose sugar, which is attached to a phosphate group to form a nucleotide. In DNA, a nucleotide is any of the four nitrogenous bases attached to a deoxyribose sugar, which, as explained, is attached to a phosphate group. The deoxyribose sugar can bond with a phosphate group from another nucleotide to form a chain. The nitrogenous base portion of the nucleotide can bond with the nitrogenous base from another nucleotide.
Nucleotides attach side by side to make long strands of DNA. They are able to attach in this fashion by the phosphate group of one nucleotide to the deoxyribose sugar of another nucleotide. This strand is formed in what is known as the 5 prime to 3 prime direction and opposite of this strand is a complementary chain which goes in the 3 prime to 5 prime direction. Therefore if the original strand is. When Watson and Crick proposed the structure of the DNA molecule, they stated that the molecule was a double helix held together by ladderlike projections.
The backbone of the helix is the deoxyribose sugar and phosphate group. One of the phenomenal characteristics of the DNA molecule is that it not only stores genetic information but replicates itself. This process is simply known as replication. Replication starts when the double strand is opened up by a helicase enzyme, which exposes the base sequences. While the base pairs are exposed along the template strand, a new strand of DNA a complementary strand is synthesized by DNA polymerase.
Replication occurs continuously from the origin of one strand, called the leading strand, which follows the 3 prime to 5 prime direction. These fragments are linked together by DNA ligase. The leading strand replicates a complementary strand with DNA polymerase delta, while the lagging strand makes a complementary strand using DNA polymerase alpha.
The process of replication results in two identical copies called daughter duplexes of the original strand of DNA. Each daughter duplex contains one parental strand from the original DNA molecule and one newly synthesized strand. This is known as a semiconservative model.
In , Matthew Meselson and Franklin Stahl used a scientific technique with radio-labeled nitrogen bases to prove that the DNA molecule replicates using a semiconservative model. In healthy cells, there is a set of postreplication-repair enzymes and base-mismatch proofreading systems.
These systems remove and replace mistakes made during replication e. Occasionally, a change in the nucleotide sequence takes place; this is known as a mutation. In , two scientists, Richard Roberts and Phil Sharp, discovered that there were many regions in the DNA that did not code for anything. Roberts and Sharp called these noncoding interruptions introns short for intervening sequences , and the sections that are coding regions are referred to as exons.
They also found that mRNA, which was thought to be an exact copy of a transcribed section of DNA during protein synthesis, was actually missing these intron regions. However, others believe that the extra sequences may stabilize the DNA molecule, or that the introns may be genetic remnants of evolution vestigial DNA and may have been expressed in the past but now lies dormant. In addition, it is conceivable that introns may have a function that presently eludes us. RNA ribonucleic acid is a small, single-stranded nucleic acid that is involved in protein synthesis.
At that time it was believed that only proteins could act as biological catalysts. Cech was able to prove that RNA could function as a biological catalyst as well. Currently nine types of RNA have been identified:. This is mostly because small forms of RNA can support life e. Finally, there is the possibility that both molecules arose at the same time, forming a symbiotic relationship. Transcription is the process by which DNA makes a copy of a section of itself; that copy is RNA and is used for protein synthesis.
In the DNA molecule, there are genes known as structural genes that code for proteins. Transcription begins when protein transcription factors attach to a promoter site on the DNA molecule. The complex of transcription factors and RNA polymerase Pol II move downstream 3 prime to 5 prime along the template strand of the DNA, unzipping it as it moves forward and reconnecting the back portion of the double helix, and forming what is called a transcription bubble.
The new mRNA molecule is then transported from the nucleus to the cytoplasm, where it will be used to make a peptide or peptides, which are used to make proteins and enzymes. On a strand of mRNA, nucleotides pair up in sets of three e. Each codon corresponds to an amino acid e.
There are 64 possible combinations of codons, but several codons represent the same amino acid e. The codons make up what is known as the genetic code. It works on the basis of tRNA, which contains and anticodon on one end and an amino acid on the other end. Translation takes place in the cytoplasm within the endoplasmic reticulum. This process takes place in three main steps:.
After translation is completed, posttranslational modification occurs, which involves the removal of methionine and peptide cleavage. DNA sequencing is a scientific method for determining the order of the nucleotide bases in an unknown strand of DNA.
The original method was devised in the early s by Walter Gilbert and Allan Maxam, and called MaxamGilbert sequencing. Their method was very labor-intensive and involved the use of hazardous chemicals. In , Frederick Sanger developed a quicker, more reliable, and less hazardous method of DNA sequencing called the Sanger method or chain-termination method.
Because they lack a hydroxyl group, they interrupt and stop the normal sequence being produced in the complementary strand from the template DNA, which causes a termination in the chain. This method is sometimes called the dideoxynucleotide DNA sequencing method, or chaintermination sequencing. The single-stranded DNA of unknown sequence is used as the template and a complementary strand is made using radioactively labeled nucleotides.
Next, the radioactively labeled complementary strand is placed in four separated mixes, each containing DNA polymerase and one of each of the four dideoxynucleotides. The four separate mixes are then run through a polyacrylamide of gel electrophoresis in four separate rows, which separates the small fragments of DNA. These four rows of fragments correspond to the particular dideoxynucleotide used. From this, a deduced sequence of the original template strand can be made.
Currently a method using automated sequencing, which uses fluorescent markers instead of radioactively labeled markers, is used. Without this technology, the Human Genome Project would have taken several decades to complete or may have even been unattainable. DNA sequencing also has applications in diagnostic testing and forensics. It can also be used to identify a specific pathogenic mutation that causes a particular genetic disease.
It had originated as an international project initiated in by the U. Department of Energy and the National Institute of Health. This project had six major goals:. The completion of the HGP is significant for the field of anthropology because it will improve the study of topics such as germ-line mutations and assist in determining our genetic relationship with Cro-Magnons and Neanderthals, as well as establish the relationship between those two species.
With the completion of the HGP, there are many anticipated improvements in anthropology, medicine, energy, and the environment. However, many ethical and legal concerns will arise as well. The first experiments involving genetic-engineering techniques were made possible by seminal works of three individuals: Paul Berg, Stanely Cohen, and Herbert Boyer.
All three scientists were separately working on research and experiments involving DNA. Eventually, they collaborated, using all of their techniques to coordinate the very first experiments involving removal of a gene from one species and inserting it into the genome of another species. During the years to , Paul Berg at Stanford University was the first scientist to complete a successful gene splicing experiment.
This research involved the removal of a gene from a viral genome called Simian Virus 40 SV40 , which was a monkey virus. He was initially interested in studying a particular gene because he found that SV40 could cause cancer in mice. The advantage of studying a viral genome was that it was very small— approximately a few hundred genes. This allowed him to easily identify and isolate this gene.
He then attached this gene to the DNA of a lambda virus known as a biological vector , which would then insert this gene into another cell. This was the very first time that a gene or genetic material from one organism, in this case a virus, was removed and spliced into the DNA of another organism, in this case a second virus.
The recombinant DNA can then function normally, replicating itself and producing its sequenced products as all other DNA normally does. Stanely Cohen was another scientist at Stanford University. His initial research was investigating how the genes in plasmids could make bacteria develop resistance to antibiotics. He implemented techniques allowing him to remove a plasmid, which was a small ring of DNA, from one bacterium and insert it into another bacterium.
Once the plasmid was inside the other bacterium it could then produce the products that it normally made in the original bacterium. This process happens naturally between bacteria and was originally observed by Fredrick Griffith, who called it called transformation. However, Cohen was able to intentionally and selectively make this process take place. Herbert Boyer, a scientist at the University of California, was doing research on a bacterium called Escherichia coli or E.
It was discovered that bacteria produce RE to defend themselves against viruses, which work by snipping viral DNA into smaller pieces rendering the virus ineffective. Today there are over RE that are used in laboratories for gene splicing and the production of recombinant DNA. After this technique was refined, Boyer later went on to genetically engineer human insulin, which was the first genetically engineered product approved by the FDA in In , the first animal gene was cloned, using the techniques refined by Berg, Cohen, and Boyer.
In a basic sense, the frog gene was removed using RE, then spliced into a plasmid, and then inserted into E. After the resulting DNA was inserted into E. The transfer of DNA from one organism into another organism is possible because DNA is universal among all organisms and cells on this planet.
The organism E. It is a very complicated process, but a simplification has been made here in order to establish an understanding of the process. This is done using restriction enzymes, which cut up the DNA into fragments. The restriction enzymes are very specific and cut the DNA at very specific points. Therefore, the DNA of interest can be located and removed. After the desired section of DNA is isolated, it then needs to be amplified because the amount originally acquired is usually not enough to be effectively transferred.
Finally, the isolated and amplified DNA needs to be introduced into the host cell. This is accomplished with biological vectors and nonbiological vectors. Biological vectors are either plasmids or viruses, which were used in the original genetic engineering experiments by Berg and colleagues. Nonbiological vectors include electrochemical poration, biolistics, microinjections, and recombinasemediated cassette exchange RMCE.
As mentioned, the DNA in all organisms and cells is made out of the same material nucleotides and sugar phosphates. There are two types of genetic modifications; one involves the addition of genetic material and the other involves the deletion of genetic material or the products it expresses. Deletion is done in one of two ways: knockout and antisense genes. In addition to modifying germ cells and somatic cells, the techniques of genetic engineering can also be used to genetically clone species.
Cloning is when two and sometimes more individuals or cells are produced from one genome. On July 5, , two scientists, Ian Wilmut and Keith Campbell, cloned the first animal from an adult somatic cell by using a technique called nuclear transfer. Other projected use of genetic engineering is the possibility of individualized or genetic medicine.
Individualized medicine is a futuristic style of medicine in which treatment will be tailored to the unique genetic needs of the patient. This is also known as personalized medicine. There are two major fields involved with the development of individualized medicine—pharmacogenetics and pharmacogenomics. Pharmacogenetics is an aspect of genetic medicine that studies the genetic sensitivity and differential response of a medication for a patient population.
Pharmacogenomics is another aspect that is geared toward the manufacturing of pharmaceuticals with methods of genetic engineering. In the near future, these two fields will change the way medicine is practiced. It is conceivable that during a typical office visit less time could be spent on deciphering somatic complaints and performing a physical exam, and more time on examining the genetics of the patient. This form of consciousness would of course be very different from neurological consciousness or human consciousness.
In fact, DNA consciousness may underlie our very own conscious process. This is a realistic possibility considering certain families of gene clusters; Hox and Pax genes are responsible for and oversee the development of our neurological consciousness. If those genes are altered or deleted, neurological consciousness does not develop. Other ideas that support DNA consciousness are that the DNA molecule replicates itself, produces proteins freely, communicates chemically with other parts of the cell, and interacts with the external environment of the cell.
It performs all of these functions independently. In addition, it is the first known molecule to discover itself i. Genetic-engineering techniques may help us to explore this area by enabling scientists to explore how DNA interacts with itself, other molecules, and the environment; how it is able to freely self-replicate, and how it knows when and when not to produce certain products.
The future applications of genetic engineering are numerous indeed. Most of the immediate impact will be seen in the fields of anthropology and medicine. In medicine, there will be improvements in clinical therapeutics and individualized medicine. This will improve life spans and harness the potential to halt or reverse the aging process. In anthropology, the completion of genome projects will assist in establishing genetic relationships between humankind and other species.
In addition to studying our evolution, we could potentially control our evolution. Therefore, emerging teleology could become a reality. The potential to alter human genomes could create the first transgenic Homo sapiens and provide the appearance of new species on this planet and elsewhere, a concept known as transhumanism.
This could also give rise to new species such as Homo sapiens futurensis the human of the future as proposed by Birx or Homo sapiens genomicus the transgenic human as proposed by Grandy. It is also conceivable that genetic engineering could potentially equip our species with genes that could improve our ability to survive in outer space. This could give rise to Homo sapiens extraterrestrialis.
Not until about did Leo Kranner give a clear definition for autism, by writing Autistic Disturbances of Effective Contact, describing his research of autism cases. The paper descriptively addresses the behaviors of the children, describing them as self- satisfied, showing no apparent affection for others, oblivious to their surroundings as if they live within themselves Kranner, From this, many other.
In this paper, there is a brief overview of how HPV replicates and what happens when the cell is infected. It will also look at methods to preventing HPV infection such as vaccines, safe sex and abstinence as well as ways to treat HPV when prevention fails including wart removal and cancer treatments.
This paper will also discusses some of the signs and symptoms if any, that HPV might exhibit when infection has occurred. Possible research in the field of diagnosing HPV will. The article is a critique about Andrew Wakefields original article in about how vaccines cause autism. Within this article they make several arguments about how this is false and has no scientific research supporting it.
They did several research experiments to disprove the work of Andrew Wakefield, Neal Halsey and Jenny. Whereas a scholarly journal contain original research, data, studies and experiments. These articles are intended for professionals, scholars, or students of a specific field. I was able to find both a popular article and a journal that I found interesting, they spoke about the first malaria vaccine trail in humans.
The host ranges from plants, bacteria, animals, and virus. The scientist isolates a plasmid from a bacteria or yeast cell, then insert the modified DNA into the plasmid. Then the plasmid is inserted into an organism DNA with the use of a restrictive. Andrew Wakefield came to this conclusion based on results found in eight out of twelve children. His results were then published in a medical journal called Lancet. Andrew Wakefield condemnation of vaccination caused the public to become scared "vaccinations and Autism".
Andrew Wakefield's research was the starting point of the conspiracy theory that the measles, mumps, and rubella.
Recent breakthroughs in the understanding of the biological processes underlying cancer development have led to more effective treatment strategies and targeted therapies have irrevocably changed the treatment of cancer patients. Yet, the use of such therapies requires the biomarker testing in tumor biopsy and obtaining sufficient amount of tumor material for the analysis of somatic mutations in targetable genes is somehow challenging.
As a result, its study permits the genetic characterization of the tumor by a non-invasive procedure and solves the difficulties of conventional biopsy 4 - 6. Moreover, unlike solid biopsies, in the liquid biopsy circulating tumor DNA ctDNA can be quantified over the course of treatment, which can potentially serve to measure tumor response to treatments 7 , 8.
In such scenario, the number of samples to test is expected to increase substantially being the development of high-throughput technologies for cfDNA isolation an important challenge to address. In this way, the emergence of automated systems could improve the reproducibility and robustness of the process and facilitate the implementation of liquid biopsy in the clinical setting. Specifically, we compare the cfDNA extraction yield and fragment size of nucleid acids obtained.
Finally, we investigate whether the identification of tumor specific mutations could be affected by cfDNA isolation methodologies, by comparing mutant allele fraction MAF measurements by digital PCR dPCR in 38 cfDNA samples from cancer patients with tumors harboring driver mutations. A total of 57 samples were obtained from cancer patients that were prospectively enrolled in the study between February and June after signing the appropriate informed consent.
Information regarding demographics, clinicopathological features, and tumor mutation status was obtained from the clinical and pathology reports. All samples were processed at room temperature within 2 h from the time of blood extraction. After centrifugation, 55 plasma samples were each divided into two aliquots of 1 mL and two plasma samples were each divided into three aliquots of 1 mL Figure S1.
Hemolyzed samples were discarded for further analysis. Once thawed, a new centrifugation was performed at 5, g for 20 minutes to ensure removal of impurities in the supernatant. Chip fluorescence was read twice. The automatic call assignments for each data cluster were manually adjusted when needed.
The result of the assay is reported as the ratio of mutant DNA molecules relative to the sum of mutant and wild-type wt DNA molecules. A wt control DNA was included in every run. Qualitative variables were summarized by their frequency distribution and quantitative variables by their mean and standard deviation SD or median and interquartile range.
The nonparametric comparison of cfDNA concentration yield by different aliquots of the same plasma sample, using different methodologies, was evaluated using the Wilcoxon signed-rank test for paired data. The nonparametric comparison of cfDNA concentration yield by different clinical situations such as tumor origin lung cancer vs. Null hypothesis was rejected by a type I error minor than 0.
Relative quantification of circulating free nucleosome bound DNA fragments was expressed as the ratio between the concentration of a particular fragment and the total concentration of cfDNA in the corresponding sample. Comparison between circulating free nucleosome bound DNA fragments ratios obtained in paired samples using different methodologies was assessed by Wilcoxon signed-rank test for paired data.
For this analysis, the level of significance was adjusted to 0. Among the 57 plasma samples collected, 47 samples corresponded to lung cancer patients and ten samples corresponded to colorectal cancer. Altogether, we measured the concentration of cfDNA in a total of samples obtained from 57 plasma samples.
Overall, the results of quantitation revealed a wide range of cfDNA concentrations in the plasma of cancer patients, ranging between 0. This subset samples were obtained from lung cancer patients, clinical stage III—IV, except for one sample that corresponded to one patient diagnosed with colorectal cancer. According to our results, the median concentration yielded was 1.
Interestingly, we observed that the amount of cfDNA obtained from stage IV cancer patients was significantly higher compared to non-metastatic patients. Regarding the comparison between MR and MPC methodologies, 26 plasma samples were processed using both methodologies. These plasma samples were obtained from stage IV colorectal cancer patients and lung cancer patients with metastatic disease except for one patient that was IIIA stage.
We also investigate whether tumor location lung or colon could influence the cfDNA isolation yield. The observed size of the cfDNA fragments was approximately bp mean bp, range — bp. In 69 out of 78 cfDNA samples, we also observed a oligonucleosomal DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes were clearly visualized in the electrophoretic assay Figure 1 , suggesting an apoptotic cell death.
Therefore, RNA sequencing has been extensively used for the detection of gene fusions in tumour tissue [ , ]. The same strategy can be applied to cfRNA [ ]; however, as RNA is more unstable, systematic optimization of preanalytical processing is required to ensure reproducibility and stability. Early screening for cancer has become increasingly effective through the collection of information from multiple different biospecimens.
Cell-based methods, such as the Papanicolaou test Pap smear , have been tested for the diagnosis and screening of endometrial cancers [ , ], but have proved challenging. In ovarian cancer, for which the sensitivity of Pap smears is low, perhaps combined Pap smear and mutation analyses may provide better performance for screening to compensate for the limitations of either approach alone [ 92 , , ].
Another strategy to improve the screening performance of cfDNA-based assays is to target biomarkers that are highly specific to the tumour and nearly absent in background cfDNA, e. EBV DNA was first identified in plasma of NPC patients in [ ], and is cleared from plasma after treatment [ , ], making it an ideal biomarker [ ].
These patients were diagnosed earlier and had better 3-year progression-free survival than historical cohorts, and only one participant with a negative EBV result developed NPC a year after testing [ ]. This example demonstrates the potential of targeting highly specific tumour-derived information in cfDNA for early screening of cancer. To date, the majority of clinical applications of cfDNA analysis have been demonstrated in prenatal testing and cancer diagnostics, but it has potential in many other physiological conditions, such as trauma, stroke, sepsis, epilepsy, autoimmune diseases, and post-transplantation monitoring [ 45 — 49 , ].
In prenatal testing, multiple molecular tests have been developed for fetal aneuploidy [ — ] or single-gene disorders [ — ], with some having been clinically implemented worldwide [ — ]. In oncology, there is tremendous clinical potential for ctDNA to be incorporated in molecular diagnostics. However, before broad clinical implementation, practical considerations such as preanalytical factors and regulatory requirements must be addressed in order to achieve consistent and reproducible results.
Preanalytical factors have different effects on cfDNA yield, quality, and downstream molecular applications. One factor is the storage time between blood draw and processing to isolate plasma. Delayed processing results in blood clotting and lysis, which results in the release of large amounts of background genomic cfDNA, introducing additional challenges for identifying low-level somatic mutations [ ].
Preservation tubes, such as EDTA tubes, that prevent blood clotting and minimize the release of genomic DNA from blood cells can be used to address this issue [ ]; however, processing needs to be performed within 2—24 h [ — ], which is challenging to implement in a clinical setting. One potential solution is to use specialist blood collection tubes, which can extend storage for many days before processing [ — ].
Other preanalytical factors, such as centrifugation protocols, number of freeze—thaw cycles, and extraction methods, are also important [ , ]. Guidelines pertaining to preanalytical factors must be put in place to ensure accurate and efficient genomic profiling. The two most important initial decisions are the choice of platform and the scale of the analysis required.
The choice of clinical sequencing platform and method depends on the sensitivity, the type and complexity of the test, the expected volume of testing, the turnaround time, the costs, the laboratory infrastructure, and the computational and human resources available to validate and perform the clinical testing.
The specific requirements for validation vary according to the intended use: single-locus, low multiplex panel testing, targeted NGS, WES, or genome-wide analysis. Single-locus or low multiplex assays, such as digital PCR, allow rapid detection and quantification of recurrent hotspot mutations and monitoring of well-established resistance mutations, with a rapid turnaround time for a relatively low cost.
These assays are highly sensitive, although restricted in the number of loci assessed. Clinical validation encompasses the establishment of accuracy as compared with a gold standard, inter-assay and intra-assay reproducibility, and the establishment of sensitivity levels for each patient population. For more comprehensive assays such as targeted NGS approaches, platform selection is pivotal to all further decisions regarding testing, validation, and capability for future expansion.
Several important considerations include the size of the panel, selection of genes, depth of sequencing, coverage, sensitivity of the assay, and complexity of analytical and clinical interpretation. All of these will factor into the cost of running the assay, which, for large panels or WES, can be prohibitively high for clinical implementation. According to current guidelines for NGS testing, the laboratory should determine gene content on the basis of the available scientific evidence, clinical validity of the variants, and utility of the NGS assay [ ], with the scientific evidence being documented in the validation protocol.
Although specific guidelines for cfDNA testing are not yet established, regulatory requirements under Clinical Laboratory Improvement Amendments call for all non-FDA-approved tests to address accuracy, precision, reportable and reference ranges, analytical sensitivity, analytical specificity, and any other parameters that may be relevant to the assay performance. All clinical validations should be included in the outlined validation protocol, including the entire range of clinical samples expected for the patient population, and, if appropriate, validation of the bioinformatics pipelines.
Given the wide range of clinical platforms used for cfDNA testing, a comprehensive evaluation that would cover all of them is outside the scope of this review. The wide variability in cfDNA content among samples introduces a high degree of complexity and more potential sources of error. Assessing all potential sources of error at every level of assay design, method validation and quality control is critical to avoid potential harm to the patient being caused by both false-positive and false-negative results.
Special attention must be given to the qualification and quantification of input cfDNA. Dedicated standard operating procedures that outline the preanalytical steps for optimal collection, handling, extraction, isolation and storage of cfDNA samples must be validated with the same rigour as the analytical platform.
Cell-free nucleic acids have tremendous potential for molecular pathology in cancer. Advances in the sensitivity and specificity of cfDNA methodologies open up possibilities for the early detection of recurrence and cancer screening at asymptomatic stages. In the molecular diagnostic setting, we envision that tumour analysis will continue to play a critical role in diagnosis by revealing histological and genomic profiles. Further work needs will further define the specifications for broad clinical implementation, but, undoubtedly, cell-free nucleic acid profiling is creating a new paradigm of molecular pathology to improve cancer care through precision oncology.
DWYT is a contributor to a patent on cell-free DNA detection methodologies, and may receive royalties related to the licences of those patents to Inivata Ltd; the terms of these royalties are managed by Cancer Research Technology and Cambridge Enterprise. MFB receives research support from Illumina and is a consultant for Sequenom.
The remaining authors declare no competing financial interests. The sponsors had no involvement in the preparation of the manuscript or decision to submit. National Center for Biotechnology Information , U. J Pathol. Author manuscript; available in PMC Jul Author information Copyright and License information Disclaimer. All authors approved the final version.
Copyright notice. The publisher's final edited version of this article is available at J Pathol. See other articles in PMC that cite the published article. Abstract Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer.
Introduction Molecular pathology is critical for cancer management, owing to the expanding application of targeted treatments that are prescribed on the basis of tumour-specific mutations. Clinical applications of cfDNA Extensive research studies and trials have demonstrated the clinical applications of cfDNA profiling at multiple stages of treatment: prognosis, molecular stratification at diagnosis, detecting resistance mechanisms at relapse, and detecting minimal residual disease MRD.
Open in a separate window. Figure 1. Prognostic value cfDNA has been shown to predict prognosis and treatment response. Molecular stratification Detection of specific mutations can be used to stratify patients and guide therapy, including adjuvant therapy [ 44 , 56 ], endocrine therapy [ 57 ], and targeted therapies.
Detecting resistance mechanisms cfDNA can be used to monitor acquisition of resistance, through screening for known resistance mutations [ 86 — 94 ], or searching for novel mechanisms of resistance [ 95 — 97 ]. Cerebrospinal fluid CSF circulates throughout the central nervous system CNS , and is protected from the systemic circulation by the blood—brain barrier [ ]; as a consequence, it possesses a low background level of normal cfDNA. Other cell-free nucleic acids and strategies cfDNA methylation studies Analysis of DNA methylation can be applied to cfDNA to uncover methylation changes known to be important in cancer by the use of methylation-specific PCR [ ], microarrays [ ], or sequencing [ — ].
Early screening for cancer Early screening for cancer has become increasingly effective through the collection of information from multiple different biospecimens. Practical considerations for clinical implementation To date, the majority of clinical applications of cfDNA analysis have been demonstrated in prenatal testing and cancer diagnostics, but it has potential in many other physiological conditions, such as trauma, stroke, sepsis, epilepsy, autoimmune diseases, and post-transplantation monitoring [ 45 — 49 , ].
Preanalytical considerations Preanalytical factors have different effects on cfDNA yield, quality, and downstream molecular applications. Considerations for clinical test development The two most important initial decisions are the choice of platform and the scale of the analysis required.
Considerations for test validation and clinical implementation Although specific guidelines for cfDNA testing are not yet established, regulatory requirements under Clinical Laboratory Improvement Amendments call for all non-FDA-approved tests to address accuracy, precision, reportable and reference ranges, analytical sensitivity, analytical specificity, and any other parameters that may be relevant to the assay performance.
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