Cell free DNA(cfDNA)

Cell-free DNA (or cfDNA) refers to all non-encapsulated DNA in the blood stream.

cfDNA comprises DNA fragments that are physiologically released from apoptotic or necrotic cells into the circulation.

These cell fragments of DNA are released from various tissues of the body after cell death.

It is a composite substance of extracellular DNA molecules found in bodily fluids, including plasma and urine.

It is made up of DNA molecules released from various tissues in the body, providing a source of non-invasive sampling that provides insight into physiologic and pathologic processes.

Short fragments of DNA molecules accounts for the majority of cfDNA, and fragmentation patterns of cfDNA carry diagnostic information:the process is referred to as fragmetomics.

The epigenetic signature of the tissue of origin is carried by cfDNA.

Information gathered by cfDNA analysis, includes methylation status, aberrant fragmentation patterns, and the presence or absence of somatic pathogenic variants in the genes APC and KRAS.

In patients without underlying malignancy, this DNA derives predominantly from apoptotic hematopoietic cells.

ctDNA analysis can be tumor informed or tumor agnostic.

Tumor informed assays detect the presence of known genetic mutations from a patient’s tumor.

Studies have demonstrated that ctDNA sensitivities of 48 to 100%, specificities of greater than 90%, positive predictive value near 100% for detecting relapse, with median lead times of detecting radiographic recurrence of 4 to 10 months.

It is typically composed of fragments that are approximately 167 base pairs.

The proportion of cfDNA derive from the tumor tissue from tumor tissue specifically called circulating tumor DNA (ctDNA) is composed of shorter fragments of 145 base pairs.

These fragments are rapidly cleared from the circulation by nucleases, renal excretion, and uptake by the liver and spleen.

Higher levels of cfDNA  may be observed in patients with cancer and non-malignant condition such as infection or inflammation.

False positive testing may be a result of the detection of somatic mutations from normal tissues, benign diseases, and clonal hematopoiesis.

The use of DNA methylation procedures can reduce above false positive findings.

cfDNA originates primarily from circulating cells, certain cfDNA molecules arise from deep tissues and can be found in plasma.

CtDNA fragmentation leads to the formation of characteristic signatures based on the tissue of origin and is determined by nucleosomal organization, gene expression, chromatin structure, and the nucleus content in the tissue of origin.

ctDNA  is cancer derived from cfDNA harboring tumor specific mutations in epigenetic modifications.

ctDNA levels can range from less than 0.1% to more than 90% of cfDNA, depending on stage, anatomical, location, and histology.

ctDNA is cleared by excretion or reuptake by organs, such as the liver, spleen, kidneys, phagocytic clearance, or enzymatic degradation by deoxyribonuclease, all of which render its half life to less than two hours.

These ctDNA fragments can be found in bodily fluids in patients with underlying malignancies,  such as the plasma, saliva, sputum, stew, urine, and CSF in patients with primary CNS tumors.

cfDNA are nucleic acid fragments that enter the bloodstream during apoptosis or necrosis.

A portion of cell-free DNA can originate from a tumor cell and is called circulating tumor DNA (or ctDNA).

ct DNA originates exclusively from tumoral tissue,  both primary tumoral site and metastases.

ctDNA fraction fluctuates during according to stage and tumor type.

Normally, such fragments are cleaned up by macrophages.

Circulating tumor DNA (ctDNA) status adds to the performance traditional risk factors, including the pathological stage, in stratifying patient’s risk for recurrence and need for adjuvant therapy.

ctDNA fragments carry all types of genetic aberrations including single nucleotide variance, insertions/dilations, translocations from the cell of origin in this constitute a highly specific marker of cancer.

ctDNA has shown that biopsy free cancer detection as well as prognostic predictive biomarkers for solid tumors have great promise.

Circulating free DNA( fDNA) has a short half-life in the circulation of approximately four minutes to 2 hours.

Patient should not be on treatment to prevent treatment from affecting the results of a liquid biopsy, and to provide the best possible chance of detecting genomic alterations.

The overproduction of cells in cancer leaves more of the cfDNA behind.

These fragments average around 170 bases in length, have a half-life of about two hours.

These cfDNA fragments are present in both early and late stage disease in many common tumors.

cfDNA concentration varies greatly, occurring at between 1 and 100,000 fragments per millilitres of plasma.

The detection of circulating tumor DNA (ctDNA) could give patients who have been treated for their early breast cancer (BC) warning of any recurrence many months before it could be detected clinically or symptomatically (Dicosmo).

Circulating tumor DNA is able to identify a patient who is going to relapse at the locoregional level.

Circulating tumor cells (CTCs) and ctDNA could potentially identify patients who could be spared further chemotherapy.

Circulating tumor DNA positivity preceded radiological or clinical evidence of recurrence in colorectal cancer patients by a median of three months ( Wang Y).

A ctDNA guided approach to the treatment of stage II colon cancer reduced the adjuvant chemotherapy use without compromising recurrence free survival (Tie J).

cfDNA is present in the plasma of individuals with breast cancer and can be used to detect patients pre-treatment with localized disease and has a sensitivity at 80% and 97% specificity.

ctDNA have identified 98% of mutations detected by coincident tissue biopsies in breast cancer.

ctDNA can detect very low MRD levels of DNA in breast cancer.

MRD at baseline is prognostic and correlates with risk for recurrence and with survival with breast cancer.

MRD monitoring by ctDNA can anticipate clinical recurrence for up to about three years in breast cancer.

ct DNA is a reliable, non-invasive method of identifying action variance for targeted therapies, as well as genomic alterations, targeted by drugs.

ctDNA can detect emergent genomics variants  that are known to cause treatment resistance.

With neoadjuvant chemo therapy breast  cfDNA  levels decreased dramatically and persistence at the end of chemotherapy reflects the existence of residual disease, making it a powerful marker for the detection and monitoring of breast cancer.

ctDNA variations are highly specific markers of tumor dynamics and could be used for monitoring minimal residual disease.

ctDNA liquid biopsies have the advantage of avoiding surgical procedures.

ctDNA Has prognostic value and studies in patients with a broad range of malignant neoplasms show an inverse relationship between ctDNA levels and survival.

cfDNA concentrations are low typically in the range from 1 to 10 ng/mL in plasma and cf DNA molecules have a short half-life estimated at less than 2.5 hours.

CSF ctDNA had a greater ability than plasma ctDNA to comprehensively represent the mutational landscape of brain metastases with CSF ctDNA detecting all BM mutations in 83.33% of patients, while plasma ctDNA was only 27.78%. 

CSF ctDNA is superior to plasma ctDNA in accurately representing the profiling of single BM. 

Plasma ctDNA could be an alternative liquid biopsy material to be applied in multiple brain metastatic NSCLC.

ctDNA presence prior to the initiation of neoadjuvant therapy is associated with worse disease free survival in patients with early breast cancer, with shorter overall survival in patients  with locally advanced rectal cancer.

ctDNA following tumor resection is associated with increased risk of relapse.

The use of ctDNA in  Stage II colon cancer has been shown to reduce the use of adjuvant chemotherapy without compromising recurrence risk.

ctDNA levels are generally higher in patients with metastatic disease compared with early cancer, and is indicative of total body disease burden.

ctDNA in patients with primary and secondary CNS tumors, the yield of plasma ctDNA is much lower than in patients with non-CNS malignancies, and is detectable only in a small subset of patients with widely disseminated disease.

Studies have shown both in the adult and pediatric population that CSF ctDNA is useful as a tumor diagnostic, molecular classification, and response monitor.

CSF ctDNA detection is associated with tumor proximity to the CSF space, tumor, burden, active disease, and leptomeningeal spread of disease.

ctDNA can detect very low MRD levels of DNA in breast cancer.

MRD at baseline is prognostic and correlates with risk for recurrence and with survival with breast cancer.

MRD monitoring by ctDNA can anticipate clinical recurrence for up to about three years in breast cancer.

In an average risk screening population, cfDNA blood tests had an 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced pre-cancerous lesions (Chung D).

ctDNA can be used in gastrointestinal malignancies to assess treatment responses and/or observe as a harbinger for emerging resistance in advance disease.

G.I. cancer patients who have a decline in ctDNA of at least 30% at four and eight weeks after initiation of systemic therapy have a longer progression free survival than  patients who did not meet this threshold.

in pancreatic duct adenocarcinoma the number of plasma alterations detected are prognostic for progression free survival come overall survival, KRAS, and TP 53 ctDNA kinetics are predictive of treatment response and that ctDNA outperforms CA, 19–9 as a predictor of early treatment response and tumor progression.

ctDNA is useful in predicting immunotherapy response.







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