Report on Cell Free DNA (cfDNA)

Early discovery and applications of Cell Free DNA (cfDNA)

In this article the occurrence of DNA which is cell free and has been presented in plasma of blood had been first discover by Metais and Mandel. After seventeen years, in the era of 1965, bendich and some of the staff hypothesized that cancer has been obtained from cfDNA which will be involved in metastasis. Moreover, it takes one more year to explore the initial association to the sickness. As well as in the era of 1966 in which Tan and some members viewed high levels of circulating cell-free DNA with in the blood of operational lupus erythematous patients. After the passage of eleven years in the era of 1977, where Leon and members utilized radio-immunochemistry to reveal that for almost half of the cancer patients the level of cfDNA within the blood mainly increased then the normal control of subject. In this author take account that patients who are suffering from metastatic cancer was mainly higher than cfDNA level in the blood. The reason behind this some of the technological restrictions. It mainly takes more 12 years for the primary experimental suggestion to support that the presence of cfDNA with in the cancer patient did certainly holds tumor DNA which has been based on temperature consistency measurement.

Moreover, some of the technological advancement has been made of the 1990 fuelled by the individuals Genome project sanction more diversive protest for the origin of tumor for the recent cancer patients cfDNA. In the era of 1994 two groups has been described the attendance of specific tumor mutations within the cfDNA. In this regard both of the groups utilized specific mutation premier to enable the intensification of PCR of significant tumor mutation in the samples of plasma of patients along with pancreatic adenocarcinoma and acute myelogenous leukemia significantly. This aspect of detection of the significant prior which has been known as mutation within the cfDNA had to become the prior method of cfDNA and studies unless and until the beginning of enormously parallel sequencing. As in this consequence that mutation specific PCR had not only be available in technology which will not be give appropriate specification for the evaluation of the weak signals of tumor. It had also been documented as in such of the pioneering studies that the assessment of tumor DNA circulation gives the tremendous applications for some of the clinical translations which has been made for diagnosis as well as the evaluation response regarding to the treatment and probable prognosis. Soon after this the primary breakthrough significantly all other kinds of tumor which and is mainly related with DNA modifications which had been explored in cfDNA such kind of variations which has been made within the status of microsatellite developers which mainly involves the loss of heterozygosity and gene intensification. As well as the occurrence of oncogenic viral DNA and some of the hypermethylation of the promotor areas of destruction of tumors genes. Furthermore, some of the early observations has been highlighted in this for the usage of cfDNA as in the form of noninvolved aspect to analyze the tumor genomes.  

In this it has also been mentioned that the chimeric state of cfDNA, and the occurrence of both tumor and normal DNA with in the Plasma of blood, which sanction the growth of implementation in other fields. Moreover, these has been obtained as in the exterior of the scope of the latest review and would only be described and stated here. Perhaps the most successful implementation of cfDNA studies had been the exploration of the enhanced admixture of fetal-derived cfDNA in mother’s blood stream which has been done by lo and his staff members. After some while same group explains that 70% of women manner male fetuses, fetal-Y chromosome classifications would be detected with in just 10mL of blood plasma. This exploration opened up a new avenue for the growth of fetal cfDNA which has been based on prenatal genetic testing. it has been explained through example, in one of the latest great study includes the 1914 women across 21 U.S. centers it had been presented that cfDNA based prenatal testing of for the aneuploidy fetal is mainly decreased false positive rate for the evaluation of trisomy 3 and 21 times lessen and specifically increased positive and negative probable values. Some of the other interesting implementation of detection which has been based on cfDNA of foreign, DNA is observing the status of hard organ transplant. As in the often release of DNA within the blood as the outcome of death of cell. The level of the donor of DNA with in the receiver blood could be utilized as the maker of rejection. Later on, some more researches have been made in this regard which has been associated with cfDNA levels which has been correlated along with the length of staying at hospital and some of the burning areas and the number of tasks has been required for scalds. The plasma level of cfDNA also correlated with required patient ventilation in ICU. Increased concentrations of cfDNA in blood had been found to be a predictor of death for the patient of intensive care unit. As well as line with these outcomes, cfDNA levels in blood has been turned out to be increased and have specific probable value for septic and sepsis shock, as well as uninfected tenderness, myocardial violation and stroke involved patient along with negative neuroimaging consequences. Whereas, cfDNA concentrations has been viewed to probable poststroke morbidity and ethics in patients with negative neuroimaging and some of the sickle cell disease. In short, cfDNA concentration has been raised in situations which includes enhanced rates of cell of death and necrosis.

Properties of Cell Free DNA (cfDNA)

 Features of cfDNA is double stranded and highly disjointed along with is most of the molecules being properly 150bp in its length. These matches wit the length of DNA which has been employed by a nucleosome, the initial unit for spatial management of DNA with in the nucleus. Furthermore, the other fragment length peaks along with the linear advancement of nucleosome units. Moreover, interestingly, there is still disagreement whether the expand and reduced honesty cfDNA which has been linked with cancer. Within the year of 2003 is the wang and members has been reported that the judgement of the related with the amount of 100 and 400 PCR products of the b-actin gene has been explained and enhanced the integrity of cfDNA within the 61 patients. These visions have been supported and by some studies which has been involved by authors and members as well, and it has been evaluated that the reduction cfDNA integrity which has been correlated with severe findings. Some of the philosophers considered that the reduced cfDNA integrity will be applied expanded. It was noted by many researchers that the decrease in the level of integrity of cfDNA can imply higher rate of apoptosis, and then higher apoptosis can corelate with increase in tumor proliferation. Then it proves that it was apoptotic and not necrotic cells that are responsible for the main source of cfDNA in the blood of cancer patient. This supposition also concludes that tumor which have higher level of proliferation can yield higher level of cfDNA in line with well-established link between cell proliferation and apoptosis rates. It was proved by many of the experimental data and the studies that 90% of the total cfDNA is in low molecular weight band (!150– 180 bp). But the amount of cfDNA in patients having cancer can differ in amount. In the review of Fleischhacker and Schmidt in 2008, they collaborate the results of 34 studies that include healthy as well as patient as subjects who have both malignant and nonmalignant diseases.

But it can be seen that the trend of the concentration of the DNA in the cancer patient’s blood is much more higher than in the blood on nonmalignant or healthy person, they have much more clear blood, so the concentration of cfDNA can differ widely and is less than 100 ng/ mL for the majority of reported cancer patients. . This is in line with our own data—in a group of 62 castrate-resistant metastatic prostate cancer (mCRPC) patients the mean cfDNA concentration was 53 ng/mL of blood. There is another way to look at these numbers by calculating the number of genome equivalent in the identified blood. Many of the cancer patients have 17000 genome equivalent per mL of the blood, in case of assuming 6pg of the DNA per diploid human genome. From the recent studies, it has been seen that on average!9,000 genome equivalents per mL of blood. The level of DNA in the blood of cancer patients can be recognized varyingly because the tumor cells from where they are extracted also vary. Jahr and his staff in 2001 published their first attempt where they estimated the level pf proportion in circulating tumor DNA (ctDNA) to the total of cfDNA. This ratio between ctDNA/cfDNA id examined by quantifying the concentration of hypermethylated CDKN2A promoter that the researcher thought to have the specifications of tumor. Hypermethylation of the CDKN2A promoter was detected in 11 of 25 specimens, "in line with previous studies," and in all those 6 cases where there is availability of both tumor tissue as well as the cfDNA, there was concordant results for methylation-specific PCR. The proportion of tumor-specific hypermethylated CDKN2A sequences ranged from 90% of the total cfDNA. After 4 years, Diehl with his staff gave the study in which they report that in 33 colorectal cancer patients, the tumor amount in cfDNA ranged about 0.01% to 1.7%. Moreover, they also write that the percentage of mutant molecules of APC gene can increase through 5-20 folds if we decrease the size of fragment in PCR from 1296-100bp. There were a lot more studies that prove this further. Like, amplicon sequencing of the PIK3CA and TP53 genes and digital PCR for the recognized structural variations and the point mutation gave chance to Dawson and his staff to examine that metastatic breast cancer by ctDNA is explained as the fraction of somatic mutant allele comprised a median of 4% of total cfDNA. At the given content of cfDNA, we expect that many of the cancer patients will have less than 3500 tumor genomes as per mL of the blood. Toward the mPCR patients, the yield of tumor DNA was about 135 genome equivalents per milliliter of the blood. Eventually, the rapid turnover of cfDNA is its one of the most important character. Tsumita and Iwanaga gave their first report I the kinetics of foreign DNA clearance from the blood of animal in 1963. They used the tritiated DNA that was injected in the blood of mice in order to prove that 99% of the radioactivity was cleared from the blood in about 30 minutes. They reported that there was high level of increase in the kidney radioactivity, then in liver and spleen that prove the renal clearing of cfDNA. Lo with his staff in 1999 focus on measuring the half life of cfDNA in the blood of mother’s postpartum. They use the real time quantitative PCR for the SRY gene. It was reported that 16.3 minutes is the half life of fetal DNA in the postpartum of the women. And there can no male derived cfDNA detected in the mother’s blood after the time of 2 hours of the birth. This result was very much similar to other original 1963 observation. There were many other studies that proved the very short half life cfDNA. Like, Fatouros and his colleagues focus on measuring the kinetics of the concentration of cfDNA in those players or athletes who just had very hard exercises and they proved that the level increase by 15 folds after exercise and stabilize by 13 folds after half an hour of exercise and then after more 30 minutes it came to the normal position. It is concluded that there are no arguments on the kinetics of the clearance of cfDNA from the blood stream but still there are no big studies on the mechanism of clearance in detail.