As a patient i would want to know asap so i could try something else. Its a terrible situation yet knowledge is power and in the case if cancer time !!! Time is everything in critical care.
Not sure of source. Found in good awhile back. Should be easy to locate. Glad u found it informative. Knowledge is power when investing in bio space.
look at Volume is that showing your conviction ? its all about volume.. typically this dip would have been met with huge buying . thus, the sentiment is negative. on a macro bio sentiment and especially in the sub sector of diagnostics. read the tape not the tea leaves. i'm stuck in this position now. hoping we get some good news !! soon.........
ED you should not that this board might control .000001% of float. nothing written here can move a stock on iota either way..
just look at the other diagnostic companies. their is no real buying. other then ILMN which is a beast. that tells me longs are giving up. when you see a huge sell off being met with 3 x normal buy side volume that indicates enthusiasm. just saying .. been trading for over 20 yrs. you can check me out at tracking the insiders dotcom. that's me in vid.
Why do you engage with such hostility. the retail sentiment is very low across the BIO space and especially in the diagnostic space. from my reading on the LION and TWITS it seems like longs are giving up. I believe this to be a contrarian indicator. neverhtess, this is my perspective. its ok not to agree yet to attack an opposing view is unnecessary. put me on ignore and have a blessed life. all good brother. I'm just a surfer in cabo , trading full time .nothing but good vibes for you and everyone. I write on the boards as I have time between trades. TROV is mid term position for me.
I stand by everything I share ! I believe character counts and that god is watching every move. I don't think there is anything wrong with bringing god into a financial discussion as Pepsi once stated as I believe everything is under his domain.
I did as I'm uncomfortable when you berate me for sharing my learnings or sentiment as my family follows. We should all be able to share without being attacked. Please ignore my posts thus eliminate your need to attack me. god bless and thanks
In 2012, Charles Swanton was forced to confront one of cancer's dirtiest tricks. When he and his team at the Cancer Research UK London Research Institute sequenced DNA from a handful of kidney tumours, they expected to find a lot of different mutations, but the breadth of genetic diversity within even a single tumour shocked them. Cells from one end differed from those at the other and only one-third of the mutations were shared throughout the whole mass. Secondary tumours that had spread and taken root elsewhere in the patients' bodies were different again1.
The results confirmed that the standard prognostic procedure for cancer, the tissue biopsy, is woefully inadequate — like trying to gauge a nation's behaviour by surveying a single street. A biopsy could miss mutations just centimetres away that might radically change a person's chances for survival. And although biopsies can provide data about specific mutations that might make a tumour vulnerable to targeted therapies, that information is static and bound to become inaccurate as the cancer evolves.
Swanton and his team laid bare a diversity that seemed insurmountable. “I am still quite depressed about it, if I'm honest,” he says. “And if we had higher-resolution assays, the complexity would be far worse.”
But researchers have found ways to get a richer view of a patient's cancer, and even track it over time. When cancer cells rupture and die, they release their contents, including circulating tumour DNA (ctDNA): genome fragments that float freely through the bloodstream. Debris from normal cells is normally mopped up and destroyed by 'cleaning cells' such as macrophages, but tumours are so large and their cells multiply so quickly that the cleaners cannot cope completely.
By developing and refining techniques for measuring and sequencing tumour DNA in the bloodstream, scientists are turning vials of blood into 'liquid biopsies' — portraits of a cancer that are much more comprehensive than the keyhole peeps that conventional biopsies provide. Taken over time, such blood samples would show clinicians whether treatments are working and whether tumours are evolving resistance.
As ever, there are caveats. Levels of ctDNA vary a lot from person to person and can be hard to detect, especially for small tumours in their early stages. And most studies so far have dealt with only handfuls or dozens of patients, with just a few types of cancer. Although the results are promising, they must be validated in larger studies before it will be clear whether ctDNA truly offers an accurate view — and, more importantly, whether it can save or improve lives. “Just monitoring your tumour isn't good enough,” says Luis Diaz, an oncologist at Johns Hopkins University in Baltimore, Maryland. “The challenge that we face is finding true utility.”
If researchers can clear those hurdles, liquid biopsies could help clinicians to make better choices for treatment and to adjust those decisions as conditions change, says Victor Velculescu, a genetic oncologist at Johns Hopkins. Moreover, the work might provide new therapeutic targets. “It will help bring personalized medicine to reality,” says Velculescu. “It's a game-changer.”
Scientists first reported finding DNA circulating in human blood in 1948 (ref. 2), and specifically in the blood of people with cancer in 1977 (ref. 3). It took another 17 years to show that this DNA bore mutations that are hallmarks of cancer — proof that it originated from the tumours4, 5.
The first practical use of circulating DNA came in another field. Dennis Lo, a chemical pathologist now at the Chinese University of Hong Kong, reasoned that if tumours could flood the blood with DNA, surely fetuses could, too. In 1997, he successfully showed that pregnant women carrying male babies had fetal Y chromosomes in their blood6. That discovery allowed doctors to check a baby's sex early in gestation without disturbing the fetus, and ultimately to screen for developmental disorders such as Down's syndrome without resorting to invasive testing. It has revolutionized the field of prenatal diagnostics (see Nature 507, 19; 2014).
“Cancer has been slower to catch on,” says Nitzan Rosenfeld, a genomicist at the Cancer Research UK Cambridge Institute. This is partly because tumour DNA is much harder to detect than fetal DNA. There is typically less of it in the blood, and the amounts are extremely variable. In people with very advanced cancers, tumours might be the source of most of the circulating DNA in the blood, but more commonly, ctDNA makes up barely 1% of the total and possibly as little as 0.01%. Early sequencing technologies were not up to the task of detecting it — at least, not consistently or reliably enough to use ctDNA as a biomarker.
“It'll help us answer questions in oncology That have never been answered before.”
But the past decade has brought sensitive techniques that can detect and quantify minute amounts of DNA. For example, an amplification method known as BEAMing — which fastens circulating DNA to magnetic beads that can then be isolated and counted — can detect ctDNA even if it is outnumbered by healthy cell DNA by a factor of 10,000 to 1.
Genetic oncologists Bert Vogelstein and Kenneth Kinzler at Johns Hopkins developed the technique, and in 2007 they described7 using it to track ctDNA in 18 people who were being treated for bowel cancer. After surgery, the patients' ctDNA levels fell by 99%, but in many cases the signal did not disappear completely. In all but one of the people with detectable ctDNA at the first follow-up appointment, the tumours eventually returned. None of the people with undetectable levels after surgery experienced a recurrence.
These results suggested that ctDNA can reveal how well a patient has responded to surgery and whether they need chemotherapy to finish off any lingering cancer cells. Researchers soon found similar results for other types of cancer. Rosenfeld and his Cancer Research UK colleagues James Brenton and Carlos Caldas showed that ctDNA provides a precise portrait of advanced ovarian and breast cancers8. And in the largest study yet, Diaz and other members of the Johns Hopkins group detected ctDNA in at least 75% of patients with advanced tumours, in organs as diverse as the pancreas, bladder, skin, stomach, oesophagus, liver and head and neck9. (Brain cancers were a notable exception, because the blood–brain barrier stops tumour DNA from reaching the bloodstream.)
Circulating DNA might perform better than the protein biomarkers that researchers have been seeking and refining for decades. Proteins are used in the clinic to diagnose illnesses and monitor people undergoing treatment. For example, prostate-specific antigen is a biomarker for prostate cancer, but it can give false positives because there are other reasons that the antigen can be elevated in the blood. False positives should be rarer with ctDNA because it is defined by mutations and other genomic changes that are hallmarks of cancer cells. And although most protein biomarkers stay in the blood for weeks, ctDNA has a half-life of less than two hours, so it gives a clearer view of a tumour's present, rather than its past. The Cambridge and Johns Hopkins teams have found that ctDNA is more sensitive than protein biomarkers when it comes to detecting breast10 and bowel9 cancers, respectively, and it is more accurate at tracking tumour disappearance, spread and recurrence.
Illustration by Oliver Munday
Both teams also showed that ctDNA was more sensitive than circulating tumour cells — intact cancer cells that also travel around the bloodstream and have been an intense area of research. In a sub-study of 16 people, Diaz's team found that where both were present, ctDNA fragments outnumbered circulating tumour cells by 50 to 1 (ref. 9). And although ctDNA was always there if the circulating cells were, 13 people with detectable tumour DNA had no trace of such cells.
But most exciting to scientists, says Diaz, is the ability to watch tumours evolve and adapt over time: “It'll help us answer questions in oncology that have never been answered before.”
For example, why do so many targeted therapies eventually fail? Gefitinib and panitumumab are among several drugs that block the epidermal growth factor receptor (EGFR), a protein involved in cell growth and division that is overactive in a number of cancers. People taking these drugs do very well — briefly. But after a few months, their cancers almost always develop resistance, often through changes to other genes, such as KRAS, which is mutated in many cancers.
To monitor patients and decide on the next course of action, clinicians would normally need to take multiple biopsies. But people with advanced cancer often have several tumours to test, and different parts of any single tumour could be resistant in different ways. Biopsies are invasive and risky, and difficult for inaccessible and fragile organs such as the lungs. “You can't just go to the patient and get five more biopsies after the treatment fails,” says Velculescu. Taking blood is simple in comparison.
In 2012, Diaz's team reported11 using ctDNA to study patients who were being treated with EGFR inhibitors. The researchers found 42 different KRAS mutations that confer resistance; on average, these turned up 5 months before imaging techniques showed that the tumours were progressing. The team was specifically looking for KRAS mutations, but Rosenfeld's group has used ctDNA to identify resistance mutations from a blind start. Last year, the researchers described how they had sequenced the complete exomes — the 1% of the genome that encodes protein — in blood samples from six people being treated for advanced breast, lung or ovarian cancers. In five cases, the unguided search revealed routes to resistance, such as mutations that prevent drugs from binding to their target proteins12.
Spotting resistance early would let clinicians take patients off toxic and expensive drugs that are unlikely to keep working. And by identifying the mutations that underlie the resistance, they could find effective alternatives or drug combinations. “The hope is that we can turn cancer from a deadly disease into a chronic one,” says Velculescu. “You treat someone with one therapy and when it stops working, you switch, or alternate back and forth.”
Despite its promise, ctDNA is not yet ready for a starring role in the clinic. For one thing, the most sensitive techniques for detecting it, such as BEAMing, rely on some knowledge of which mutations to look for. This knowledge can be provided by taking a biopsy, sequencing its mutations, designing patient-specific molecular probes that target them, and using those probes to analyse later blood samples — a laborious approach that must be repeated for each patient. The alternative is to use exome sequencing, as Rosenfeld's team did. This requires no previous knowledge about the cancer, but it is prohibitively expensive to sequence and analyse every sample at the depth required to detect rare mutant fragments.
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Maximilian Diehn, a radiation oncologist at Stanford University in California, has tried to combine the best of both worlds. His team identified a small proportion of the genome — just 0.004% — that is repeatedly mutated in lung cancers13. Whenever the researchers get a new blood sample, they sequence this fraction 10,000 times over. This picks up even rare mutant fragments, and the focused approach keeps costs down. Because almost everyone with lung cancer has at least one mutation in these regions, the method should work in almost every patient, says Diehn. The team is now working to develop similar mutation panels for other types of cancer, and to validate the technique in clinical trials — work that could take several years.
Like practically all ctDNA biopsy techniques, Diehn's approach does not do well at picking up early forms of cancer. In a small study13, it detected every lung cancer of stage II or higher, but only half of stage I tumours. This is understandable — advanced cancers simply discharge more DNA — but it limits ctDNA's potential as a cancer-screening tool.
Diehn says that more-sensitive techniques could overcome this problem, but Diaz disagrees. “The limiting factor is biology,” he says. “There just aren't a lot of fragments in circulation.” And if ctDNA hints at the presence of an undetected cancer, what then? “If you detect a mutation in the circulation, you don't know where it's coming from,” says Diaz.
There are other unknowns, too. Does ctDNA paint a truly representative portrait of a cancer? Do tumours that have spread to other organs release as much DNA as the original tumours? Do all the cells in a tumour release as much ctDNA as each other? Diaz says that the only way to answer these questions is to do 'warm autopsies' — to take samples and characterize all of a person's tumours very soon after death, and compare them with ctDNA extracted in life. “This is the heavy lifting that'll need to be done in the field,” he says.
And the biggest question remains: does an accurate picture of tumour burden, or a real-time look at emerging mutations, actually save patients or improve their quality of life? Even if doctors discover that someone's tumour has developed a resistance mutation, that insight is useless if there are no drugs that target the mutation. “The limitation is the reality of targeted therapies,” says Velculescu. “You get all this information — but so what? Our approaches to understanding cancer are outstripping our clinical options.”
Even if ctDNA does not yet affect outcomes, scientists say that it is an invaluable research tool, and clinicians are starting to collect it routinely. Swanton, for example, is leading a £14-million (US$24-million) lung-cancer study called TRACERx (Tracking Cancer Evolution Through Therapy), which will use both conventional biopsies and ctDNA collected once every three months. The circulating DNA may or may not provide clues that help the study participants, but at the very least, it will give Swanton a much better understanding of how lung cancer evolves, and how to control that evolution.
As Rosenfeld argues, it is better to have this information than not to. Currently, he says, “we're groping in the dark. Why would you do that if you have a tool that allows you to see what's happening?”
Advances in Gene Sequencing Led to Clinically Useful ctDNA Tests
A major factor in the surging interest in ctDNA has been the rapid drop in cost for analyzing genetic information. In a development that puts Moore's Law to shame, costs for whole genome sequencing dropped from $1 billion for the first complete genome sequenced by the Human Genome Project to $350,000 per genome in 2008 and more recently to about $1,000 for a genome sequenced with Illumina's commercially available HiSeq X Ten sequencing system.
Cancer researchers have been challenged by the problems of how to discriminate ctDNA from normal cell-free DNA, to detect extremely low levels of ctDNA, and to count accurately the number of mutant ctDNA fragments in a blood sample. The sensitivity of polymerase chain reaction (PCR)-based digital approaches improved with the addition of NGS, in which DNA is fragmented into small segments that can be quickly sequenced in millions of parallel reactions known as "reads" and then reassembled so that the set of reads shows the entire DNA sequence.
The half-life of ctDNA is about two hours, and changes in ctDNA levels can be apparent days to weeks before changes in imaging or in protein biomarkers. Because ctDNA is specific for the individual patient's tumor, it is likely to avoid some of the false-positive problems associated with other cancer biomarkers.
Detecting Cancer and Monitoring Cancer Stages Without Biopsy
Most ctDNA strands contain between 180 and 200 base pairs, similar to the 180 base-pair multiples characteristic of apoptosis, and are thought to result mainly from passive release into the blood of ctDNA after cell death.
The presence of ctDNA after resection (but before adjuvant chemotherapy) indicates residual disease. Absence of ctDNA might identify a patient subgroup at low risk for recurrence who could be spared the risk, expense, and discomfort of adjuvant therapy. In 2008, a team of Johns Hopkins researchers reported that mutation-specific probes for 18 subjects undergoing multimodality therapy for colorectal cancer and monitored for two to five years showed that "ctDNA measurements could be used to reliably monitor the tumor dynamics in subjects with cancer who were undergoing surgery or chemotherapy."
The authors suggested that ctDNA levels reflect the total systemic tumor burden because they decreased after complete resection and increased as new radiologically-apparent lesions developed. The researchers also pointed out that micrometastatic lesions (smaller than a few millimeters) contribute to tumor burden and to ctDNA levels although they are not detectable by imaging.
"For a melanoma patient who is free of disease after surgery but at risk for recurrence, ctDNA could be a nice way to follow without having to do frequent CT scans," Dr Chapman said. It is also expected to be useful in situations in which tissue biopsy is undesirable or cannot be done.
However, an important unanswered question is how often these tests should be done. "We need to know how meaningful small changes in the ctDNA level are — sensitivity, specificity, and lead-time bias," he said.
Monitoring Tumor Burden, Response to Therapy, and Resistance
"Another key advantage is that ctDNA could overcome the issue of tumor heterogeneity," commented Dr Park. "Different sites of disease often have different mutational profiles. Since blood, and therefore ctDNA, acts as a 'reservoir' for all sites of disease, ctDNA is representative for all sites of metastases."
A research team from Dr Chapman's lab led by Parisa Momtaz, MD, reported at this year's American Society of Clinical Oncology (ASCO) Annual Meeting that in melanoma patients with BRAF-v600E mutations, ctDNA correlated well with tumor burden measured by radiographic imaging.
"The ASCO cohort of 11 patients was to convince ourselves that we could get the assay to work (proof-of-principle) and that it correlated with what was clinically going on," Dr Chapman said. "We have now studied ctDNA in about 60 patients. We are focusing on patients treated with immunotherapy because radiographic evaluation of these responses are somewhat equivocal. We hope that ctDNA will add more clarity and tell us whether the immune system is attacking the tumor or not."
Also at ASCO, Nicholas C. Turner, MD, consultant medical oncologist at the Institute of Cancer Research, London, United Kingdom, and colleagues reported that in primary breast cancer tumor-specific ctDNA levels can predict early relapse.
ctDNA might also provide early warning that the patient has developed treatment-resistant disease. Sarah B. Goldberg, MD, MPH, assistant professor of medicine at Yale University School of Medicine, New Haven, Connecticut, and colleagues reported that ctDNA could be used to detect both sensitizing and resistance EGFR mutations in patients with advanced lung cancer treated with EGFR tyrosine kinase inhibitors. The researchers suggested that using NGS to detect sensitizing and resistance mutations in plasma ctDNA might allow earlier identification of resistance in patients treated with targeted therapies.
Similarly, a team of researchers from nine cancer centers in Italy found that KRAS mutations in ctDNA could be detected in over 35% of patients with non–small-cell lung cancer who became resistant to tyrosine kinase inhibitors.
In discussing this research, Luis A. Diaz Jr, MD, from the Ludwig Center for Cancer Genetics and Therapeutics at Johns Hopkins University School of Medicine in Baltimore, and Alberto Bardelli, PhD, from the Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, University of Torino, Italy, wrote, "This understanding of the mechanisms of acquired resistance to targeted agents at the molecular level can be used to plan combinatorial treatments with drugs that will suppress the expansion of the clones that are responsible for most of the current failures of medical treatment. This knowledge could result in the early adoption of alternate therapies before clinical resistance is detected."
Dr Chapman said, "I have this fantasy that you might use this to screen chemotherapy drugs in a patient. You could give them one dose of drug, then measure their ctDNA response. If there is no tumor death, the ctDNA levels wouldn't change."
ctDNA: From Bench to Bedside
Currently, monitoring genetic changes in a tumor requires multiple biopsies. "In the future, it might just be a matter of drawing a tube of blood," Dr Chapman said.
However, Dr Park warned that there has yet been little to no validation of ctDNA testing and that published studies show considerable variability due partly to lack of quality control and uniform standards.
"How the plasma is prepared makes a huge difference, which isn't always appreciated. How the ctDNA is analyzed is also quite variable among studies, with some technologies being better suited for specific applications. So for now, we would counsel clinicians not to jump the gun on this. We have to be extremely thoughtful and careful when dealing with ctDNA and its applications," Dr Park commented. He quoted another researcher (Dan Hayes, MD, from the University of Michigan), who says it best: "A bad test can be just as bad as a bad drug."
"Therefore I believe we need to apply the same rigorous standards of testing drugs to the development of ctDNA as a liquid biopsy," Dr Park said.
Abstract: Circulating tumor DNA (ctDNA) is now being extensively studied as it is a noninvasive “real-time” biomarker that can provide diagnostic and prognostic information before, during treatment and at progression. These include DNA mutations, epigenetic alterations and other forms of tumor-specific abnormalities such as microsatellite instability (MSI) and loss of heterozygosity (LOH). ctDNA is of great value in the process of cancer treatment. However, up to date, there is no strict standard considering the exact biomarker because the development and progression of cancer is extremely complicated. Also, results of the studies evaluating ctDNA are not consistent due to the different detection methods and processing. The major challenge is still assay sensitivity and specificity for analysis of ctDNA. This review mainly focuses on the tumor specific DNA mutations, epigenetic alterations as well as detecting methods of ctDNA. The advantages and disadvantages will also be discussed.
Keywords: Circulating tumor DNA (ctDNA); biomarker; DNA mutation; DNA methylation; cancer
Submitted Aug 18, 2015. Accepted for publication Aug 25, 2015.
Cancer is a leading cause of death globally which needs appropriate diagnosis methods. “Solid biopsies” cannot always be performed since it has invasive characteristic and cannot reflect current tumor dynamics or sensitivity to the treatment. Therefore, it is of great value to develop noninvasive detecting methods that could monitor the real-time dynamics of cancer. “Liquid biopsy” of circulating nucleotide acids [circulating tumor DNA (ctDNA), circulating RNA or microRNAs, etc.] may be an ideal one for patients with cancer (1). This review aims at describing the current contribution of ctDNA detection in cancer patients and to analyze the advantages and disadvantages.
ctDNA mutationsOther Section
Cancer cells often rely on the activation of dominant oncogenes for proliferation and survival. The presence of specific gene alteration can have diagnostic value, reflect patient’s responsiveness to the treatments and predict survival (2). Tumor DNA can be detected by tracking tumor-specific mutations or aberrant rearrangements. Since multi-site biopsies repeated sequentially is unpractical, a non-invasive DNA detection approach from peripheral blood may help reflecting dynamic changes in cancer cells. At present, advanced technologies have turned precise ctDNA mutation detection to reality (3).
Genetic mutation in cancer and genome-wide association analysis
Genetic mutation profiles
Variety types of cancer are sensitive to inhibition of a certain kind of molecular pathway signaling. Somatic mutations and epigenetic alterations in known components of the signaling network might influence treatment efficacy. Moving beyond the target gene, tumor cells can develop therapy resistance through acquisition of mutations (4). Therefore, additional pathway signatures might be involved in prediction of individual response.
Multigene test moved the clinical pharmacogenomics tests away from conventional sequencing of single genes and marked a huge step towards more comprehensive analysis of cancer genome ever since. It allows the oncologists to identify the targetable genetic aberrations and direct the patients to the targeted therapy if a potential drug is available (5,6). However, cancer is a multigene disease which arises as a result of the mutational activation of oncogenes coupled with the mutational inactivation of tumor suppressor genes. These genetic alterations can synergize or antagonize each other and often occur according to a preferred sequence.
Molecular cytogenetic expectation
The methods of molecular cytogenetic, such as spectral karyotyping, fluorescence in situ hybridization (FISH) and chromosome-based comparative genomic hybridization (CGH) improved resolution and genome coverage compared with conventional karyotyping based on the visualization of metaphase chromosomes. However, the most substantial developments in obtaining increasingly more detailed and comprehensive characterizations of tumor genomes have been described during the last decade. The emergence of a range of new technologies including “omics” profiling, microarray-based CGH and single nucleotide polymorphism (SNP) analysis, and next-generation sequencing (NGS) enabled to interrogate the tumor genome and proteome in a more unbiased way and led to remarkable insight into tumor biology (7).
Genome-wide scan using microarray platform are applied to identify genetic variants, including Array-based CGH, which is one of the approaches to improve the detection of structural variation affecting many base pairs. This technique is based on the principle of complementary hybridization between the array of oligonucleotide probes immobilized on a slide and two differentially fluorescently labeled test and reference DNA samples (8). The principles of SNP array are similar and based on the hybridization of fragmented single-stranded DNA to arrays containing unique nucleotide probe sequences immobilized on solid surface (9). The specialized equipment can measure the signal intensity associated with each probe and its target after hybridization. SNP array platforms contain oligonucleotide probes that interrogate both copy number and SNP sites. Genome-wide association studies (GWAS) became possible by the availability of chip-based microarray technologies for analysis of more than one million SNP (10,11).
Mutation detection using cutting-edge technologies
Targeted plasma re-sequencing (TAm-Seq)
Forshew et al. reported that de novo mutation can be detected through TAm-Seq noninvasively in 2012, which they termed as TAm-Seq (12). It allowed the re-sequencing of approximately 6,000 nucleotides whilst maintaining high depth analysis. The authors conducted a proof-of-concept experiment by tracking ctDNA from an ovarian patient, which had been re-sequenced tumor tissue from a right oophorectomy specimen and identified a TP53 mutation. TAm-Seq analysis revealed the emergence of an EGFR mutation in plasma samples, as the cancer progressed, which was not found in the original specimen. Further investigation identified low frequencies of EGFR mutation from initial samples. Forshew et al. hypothesized that as chemotherapy regimens restrained the growth of other clones, the resistant EGFR clone, which was initially present only at low frequency, gained in dominance. They demonstrate that plasma analysis can identify heterogeneous clones from different sites of the body.
Massively paralleled sequencing (MPS)
Personalized analysis of rearranged ends (PARE) was developed by Leary et al. to detect unselected genetic events that span across the whole genome (13). Similarly, another MPS named “Shotgun” was used by Chan et al. in 2013 (14). They identified copy number variations and single nucleotide variants (SNVs) of the whole genome from the plasma of 4 patients with hepatocellular carcinoma (HCC). Furthermore, they demonstrated the ability of MPS to track ctDNA level changes pre- and post-surgery. Interestingly, shotgun MPS of the plasma was also able to distinguish between tumor types in a patient with synchronous breast and ovarian tumors. The above studies illustrate that ctDNA analysis, through de novo mutation detection, can continue to track disease burden as tumors evolve, without the need for re-biopsy.
Whole-genome sequencing (WGS)
WGS enables detecting ctDNA in patients prohibitively expensive, regarding the limit analysis of whole genome MPS to a small number of samples due to expense (15). Although low depth, and therefore reduced cost, WGS approaches have been successful at detecting copy number variations, a higher depth of coverage is often required to detect rearrangements at high resolution or SNVs directly from plasma DNA. Furthermore, where low mutant: wild type allele frequencies exist, e.g., in early stage disease, an even higher depth of coverage would be necessary to detect ctDNA fragments. In addition, WGS approaches detect a higher ratio of intronic or passenger mutations than targeted re-sequencing (16). The clinical significance of passenger mutations is currently unknown and often not targetable.
Whole exome sequencing (WES)
To make routine analysis of de novo mutations in serial plasma samples possible, WES was performed to track tumor evolution in response to therapy. Murtaza et al. used this approach in a proof-of-concept study involving 6 patients with metastatic tumors. Plasma samples were collected at the beginning of treatment and at the time of relapse. Subsequent re-sequencing and variant analysis revealed that by comparing the relative representation of mutations in pre- and post-relapse samples, one could identify enrichment of mutations that may drive resistance WGS can screen a larger spectrum of the genome but is currently too expensive for routine use to detect SNVs, whereas WES approaches allow more in-depth interrogation of multiple regions but is less sensitive to identifying copy number changes (17). This work demonstrated a much more cost effective way for mutation sequencing.
Tumor-specific gene mutations
Pancreatic cancer has the distinction of being the first solid tumor associated with a specific mutation in ctDNA. This is partly because the KRAS gene is frequently mutated and easy to detect. Sorenson et al. used allele-specific amplification to assay for mutations in codon 12 in the plasma or serum of pancreatic adenocarcinoma patients (18).
The sensitivity of detecting primary pancreatic cancer on the basis of ctDNA is mostly 30% to 50% while the specificity is generally higher (approximately 90%) (19). A variety of detection methods including restriction digestion and single-stranded conformational polymorphism have been used. In one study, sensitivity was improved when CA199 was measured in combination with DNA measurements (20). However, although at a lower frequency (5% to 15%) than adenocarcinoma, pancreatitis cases also showed KRAS gene mutilations (21). Most studies have focused on KRAS mutations in pancreatic cancer because of their prevalence, other approaches have been tried. The advents of higher-throughput methods, such as NGS and digital PCR, have had a profound effect on this field. For instance, one recent study using this method showed that pancreatic duct cancer had a high rate of ctDNA than other malignancies, more so in metastatic disease than non-metastatic disease. In summary, for clinical and biological reasons, pancreatic cancer is an ideal candidate for the diagnostic and prognostic use of detection of ctDNA (22).
Plasma or serum mutation status of KRAS, APC, and TP53 which have a high mutation frequency with colorectal cancer is correlated with diagnosis, prognosis, and also treatment response (23,24).
The overall detection rates of KRAS mutations in serum or plasma of patients with colorectal cancer were 25% to 50% (24). Also, KRAS mutations in ctDNA have been reported to have the highest level in patients with more advanced stage (25). Besides, KRAS mutations in ctDNA are also associated with a higher risk of recurrence after surgery (26,27). Analysis of circulating mutant DNA could also monitor response to monoclonal antibody therapy for colorectal cancer, which makes repeatedly monitoring patients during treatment possible (28).
The exploration for APC mutations in ctDNA has focused on exon 15, which is a hotspot for APC mutations in colorectal cancer. The rate of APC mutation detection in primary ctDNA is approximately 45%. As for TP53, the mutation rate has been identified in ctDNA in about 40% of cases. Most studies focused on portions of TP53 between exons 4 and 8, the most commonly locations of TP53 mutations in colorectal cancer (24).
Unless targeting ctDNA alterations in hotspots of certain genes, a panel targeting mutations of the KRAS, TP53, and APC genes enabled the detection of at least one gene mutation from approximately 75% of colorectal cancer tissue. However, those mutations could only be detected in the serum of 45% of these patients (29).
ctDNA methylation—epigenetic changesOther Section
Unless DNA mutation, there is also gene methylation which affects their expression that can be found in ctDNA. Tumorigenesis is regulated not only by genetic but also by epigenetic alterations (2). In fact, as for detection, there are a variety of genes mutated in tumors, even when a gene is consistently mutated in a particular cancer, the gene mutations may be spread over large region that makes evaluation difficult. DNA methylation tends to occur in CpG dinucleotides in the promoter region of tumor suppressor genes that leads to expression silencing (30). Therefore, methylated ctDNA in recent years is becoming an emerging target and shows promising results.
Methylation detection methods
Methods for methylation detection emerge in an endless stream. Generally it is divided into 3 categories: (I) methylation content: high-performance liquid chromatography (HPLC) or high-performance capillary electrophoresis (HPCE); (II) candidate gene: methylation-sensitive restriction endonuclease-PCR/Southern (MSRE-PCR/Southern), bisulphite sequencing, methylation-specific PCR (MS-PCR), MethyLight, etc.; (III) methylation pattern and methylation profiling: restriction landmark genomic scanning (RLGS), amplification of inter-methylated sites (AIMS), Methylated CpG-island amplification (MCA) and so on. The most common method at present studies is usually MS-PCR.
Tumor-specific gene methylation
Methylation of ctDNA has been reported for many years. Detections of ctDNA methylation mostly focused on colorectal cancer, lung cancer, breast cancer, pancreatic cancer as well as some other types of carcinoma. Compared to mutations, the consistency of DNA methylation alterations makes it a potential promising biomarker for diagnosis, staging, monitoring response and predicting survival for cancer patients.
Colorectal cancer is the third most common cancer both in men and in women worldwide (31). An easy and quick screening test is essential for the early diagnosis for colorectal cancer. It is reported that the promoter hypermethylation status of SEPT9 was high associated with the development of colorectal cancer. In PCR-based retrospective trials for SEPT 9 promoter methylation, sensitivity and specificity were 72-90% and 88-90% respectively (32-35). Another research from USA showed that methylated SEPT9 DNA in plasma may help screening out 72% colorectal cancer with a high specificity of 93% (34). Furthermore, SEPT 9 methylation could be found in precancerous lesions of colorectal cancer. Church et al. conducted a large, prospective trial to assess the accuracy of circulating methylated SEPT9 DNA for detecting colorectal cancer in 7,941 patients using a commercially available assay. It showed a disappointing result that sensitivity and specificity were 48.2% and 91.5%, respectively (36). That may because the population they enrolled were not confirmed cancer patients, which differs to other researches.
Other biomarkers such as well-known novel sequences of APC, RASSF1A and E-cadherin (37,38) as well as novel markers in plasma have also been found to correlate with colorectal cancer. A German study showed that Methylation of helicase-like transcription factor (HLTF) and hyperplasic polyposis 1 (HPP1) in serum significantly correlated with tumor size, stage, and metastatic disease, and were also prognostic factors in metastasized colorectal cancer (39).
Besides, methylation status of H3 lysine 9 (H3K9me3) and H4 lysine 20 (H4K20me3), which are hallmarks of pericentric heterochromatin in healthy donors and patients with colorectal cancer was tested and showed weak correlation between cNUCs and histone methyl marks (40).
Breast cancer is the most common cancer in women both in more and less developed regions. Numerous studies were conducted aiming to analyze the methylation status of biomarker genes in breast cancer and assess possible clinical value, mostly using the candidate gene testing. That means a majority of the makers are the well-established genes, such as cyclin D2, RARβ2 (41), ESR1 (42) and so on. Dulaimi et al. have found that at least one hypermethylation of APC, RASSFIA or DAP-kinase could be found in 94% serum samples of all the breast cancer patients (43). Scholars from All India Institute of Medical Sciences have conducted a serious of prospective studies including 100 invasive ductal breast cancer patients. Methylation status of multidrug resistance 1 (MDR1), Stratifin, ERα and PR, DNA repair genes-BRCA1, MGMT and GSTP1 were tested. Significant correlation was found between methylation status of the promoter of the above genes in tumor tissue and paired serum. However, the sensitivity of these genes was not high (MDR1 50%, Stratifin 56%, ERα 55%, PRB 55%, BCRA1 22%, MGMT 26%, GSTP1 22%) (44-46).
Unlike using the candidate genes, methylation detection in 56 genes (MethDet-56) test was conducted to find novel methylated genes and assess the dynamics of methylation so as to monitor treatment (surgery and hormone therapy). Larger study based on these results was encouraged (41).
Lung cancer is the leading cause of cancer-related death partly because the absence of early detection approach (47). Up to now there is no ideal early diagnostic method. Changes in DNA methylation may occur on its early stage. DNA methylation detection is expected to be an essential method in early diagnosis of lung cancer. There are more than 80 hypermethylated genes related to lung cancer such as APC (48,49), RARb (50,51), RASSF1A (52), CDH13 (48,51), SHOX2 (53), SHP-1 (54). Unless early diagnosis, study focused on correlation of methylation and survival of lung cancer patients showed that CHFR methylation status correlated the results of second-line chemotherapy or EGFR TKIs in 179 of 366 patients (55).
Over-all, methylation test on ctDNA is a very encouraging and promising method to diagnose or monitor tumor. As a unique biomarker having the sufficient specificity and sensitivity is not available, a panel of multiple genes could be used
I just scanned form 4's via SEC website and didn't see anything ! WOW. that was desperate of you...
never said I was the smartest guy ! just ask my wife ! hahaha.
Nevertheless, Its my instinct that the market wants Ctdna testing and TROV streamlined approach to collecting , testing and providing analysis back to Docs is going to be hugely received. CTC testing might be a combo strategy either way Ctdna is here to stay. I believe TROV very methodical approach via data data and more data will when the confidence of the space ! regarding BIOC ,I just don't know enough to make or draw conclusions. I hope everyone makes money and then puts some to work in the communities that need help. PS: I'm very impressed with some of the work MYGN is doing its next level stuff.