Tumor sequencing can be accomplished because rapidly growing tissues, such as malignancies, shed their DNA content into the bloodstream due to high rate of cell death.
Liquid biopsy can be performed primarily on blood, but as well on other body fluids, such as urine, saliva, CSF, or third space effusions.
Liquid biopsy refers to the collection of bodily fluids, often blood, and the associated genetic materials RNA, DNA, and/or cells.
Liquid biopsy relies on identifying tumor markers, such as circulating, tumor cells, cell tumor DNA, cell free RNA, exosomes as well as tumor derived metabolites and proteins in biofluids.
Liquid biopsy allows for the diagnosis and analysis of cancer by sampling cancer cells or byproducts present in biological fluids such as blood or urine.
Liquid biopsy allows for genomics, profiling, capturing, temporal, spatial, molecular heterogeneity, managing tumor dynamics, studying treatment, resistance, and mechanisms, and detecting microscopic disease.
Genomic profiling via liquid biopsy.
Liquid biopsy can be based on measuring mutations from genomic circulating tumor DNA (ctDNA and can be measured on based on methylation or epigenetic signatures.
Liquid biopsy is the best available technological tool to improve clinical decision making in precision oncology.
Liquid biopsy done in healthy volunteers aged greater than 65 years show that 10% of these individuals already carry genetic aberrations.
Liquid biopsy assay assessing genomic profile of a tumor.
Liquid biopsy sequencing results, on average, are returned in as many as six days earlier than tissue sequencing.
A liquid biopsy is that it reflects the vast pool of alterations in a patient, assuming that the sample confers all available DNA.
Liquid biopsy limitations: DNA sample is small and may be prone to missing small amounts of mutant DNA, patients with limited disease burden, tumor lineages that release only small amounts of DNA into circulation, and certain alteration types, such as copy number changes, may be more difficult to identify.
Liquid biopsy advantages compared to traditional tissue biopsy include: it allows the detection of small tumors, minimal residual disease, and micro metastatic disease that cannot be detected with traditional biopsy; it detects ctDNA released into the bloodstream from multiple tumor regions and makes it possible to identify intratumoral heterogeneity as well as clonal evolution and ; can detect small quantities quantitative variations within the blood enabling real time surveillance.
A liquid biopsy can pick up genomic alterations across the entire body, as it detects DNA emitted into the bloodstream from any tumor site.
Circulating DNA, circulating tumor DNA (ctDNA): ctDNAis the fraction of cell free DNA cells released by tumor cells, following cell death, or by active secretion.
ctDNA levels depend on tumor burden and tumor shedding.
Most current liquid biopsy essays are ct DNA tests.
Up to 10-20% of tissue biopsies of malignancies are inadequate for molecular testing due to insufficient tissue or amplifiable DNA.
Tumor biopsies are limited by tumor heterogeneity, particularly in the setting of tumor resistance, and may yield false negative results.
The DNA shed by tumor cells is known as circulating cell-free DNA.
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.
Approximately 300-600 genes known to cause cancer.
Among the thousands of mutations acquired by a cancer cell, evidence suggests only a handful actually instruct the cell to function as an autonomous clone, and these are called driver mutations.
The protein coding of the human genome is only 1%, but the vast majority of driving mutations fall within this portion, with approximately 300-600 of the more than 20,000 protein coding genes being targets for driver mutations.
The remaining mutations are named passenger mutations, acquired by the clone before the first drive mutations arose during or after its subsequent transformation.
Driveway mutations include substitutions of one base of DNA for another, insertions, deletions of small numbers of DNA bases, gains and losses of large chromosomal regions or even whole chromosomes, and rearrangements that fuse one gene to another or juxtapose one gene with the regulatory apparatus of a nerve.
Because driving mutations are causative for cancer, drugs that target the function of resulting proteins can be therapeutic: Imatinib targets the BCR/ABL fusion protein in CML, BRAF inhibitors for BRAF mutant melanoma, EGFR inhibitors for non-small cell lung cancers, Anaplastic lymphoma kinase inhibitors for ALK lung cancers, and anti-human epidermal growth factor receptor 2 antibodies for HER2 amplified breast cancers.
ctDNA have identified 98% of mutations detected by coincident tissue biopsies 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.
Somatic mutations can arise due to endogenous in exogenous mutational processes.
Exogenous mutagens include chemicals such as tobacco, aflatoxin B1, and chemotherapy agents, ionized radiation and ultraviolet light.
All of the above damage DNA, generating mutations when damage bases are incorrectly repaired or copied.
Mutations can also arise from cell intrinsic processes, such as errors that occurred during DNA replication, reactive oxygen species, impaired DNA repair, and activity of viruses. Many intrinsic cell processes occur at a constant rate throughout life, leading to linear accumulation of mutations with increasing age.
Allows the ability to biopsy metastatic disease that has infiltrated difficult to reach sites, such as the brain, bones, and liver.
Allows for the longitudinal study of tumors.
Unlike genomic DNA, tumor cell DNA (ctDNA) is highly fragmented.
Most ctDNA fragments are approximately 150 bp in length.
cfDNA exists in low concentrations, with absolute level of cfDNA levels are less than 100ng/mL, and only a fraction of this total cfDNA is actually tumor derived.
cfDNA for various cancers at low levels are associated with a more favorable outcome than high levels.
ctDNA levels correlate with mitotic rate, cell death,tumor vascularization, extent of metastatic disease burden and sites of metastases.
ctDNA can be used for the detection of minimal residual disease particularly after surgical therapy to remove primary tumors, as a predictive residual disease and relapse and as in indicator of poor prognosis.
Liver and bone metastases are associated with higher levels of ctDNA.
Normally very significant amounts of circulating cell-free DNA most of which is derived from hematopoietic and epithelial tissues.
In the presence of a malignancy there is only a tiny percentage of cell-free DNA that is tumor derived.
Cell free DNA(ctDNA) is thought to be released by tumor cell apoptosis, necrosis, and extracellular vesicle secretion.
Plasma genotyping can non-invasively and rapidly detect and monitor genomic alterations over time, and has the potential form overcoming limitations of tissue biopsy.
DNA not detectable in 5-15% of patients with advanced cancer.
By capturing tumor-derived cell-free DNA fragments the tissue can be sequenced and the genome reconstructed without the need of a tissue biopsy.
There is greater than 100 times more tumor-derived material in the cell-free domain than in the cellular domain.
The higher amount of material in the cell-free domain allows a better capture of tumor heterogeneity and potential for earlier detection of relapse and treatment failures.
Cell-free DNA fragments have been detected in virtually every malignancy, with primary brain tumors being an outlier due to the blood brain barrier.
Guardant360 measures circulating DNA.
Gene panel includes: ALK, APC, AR, BRAF, CDKN2A, EGFR, ERRB2, FBXW7, KRAS, MET, MYC, NOTCH1, NRAS, P1K3CA, PTEN, PROC, RB1, TP53,
ABL1,AKT1, ATM, CDH1, CSF1R, CTNNB1, ERBB4, EXH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HTAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TERT, VHL.
Circulating tumor DNA (ctDNA) levels before and during treatment can predict how a patient with diffuse large B cell lymphoma responds to standard therapy.
Long-term outcomes can be predicted as early as three weeks after iniation of drug therapy.
A liquid biopsy test called Guardant360 appears to be comparable to standard tissue biopsies in detecting guideline-recommended biomarkers in advanced non-small-cell lung cancer (NSCLC).
In NSCLC liquid biopsy has a role in monitoring response to treatment, detecting residual disease or early recurrence, and detecting early stage cancer.
It also has a faster turnaround time and may identify more patients who can be treated with targeted therapy.
The Guardant360 test detected 7 known predictive biomarkers, including genomic alterations in ROS1, BRAF, RET, MET, ALK, EGFR, and ERBB2, as well as 1 prognostic biomarker, KRAS mutations.
The detection rate of targetable mutations using Guardant360 liquid biopsy is similar to the detection rate of standard of care testing of tumor tissue.
DNA sequencing refers to the process of determining the nucleic acid sequence, the order of nucleotides in DNA.
In contrast to adult malignancies, which are frequently driven by oncogenic mutations, many childhood cancers have a low burden of somatic mutations and have a much higher likelihood of being caused by her line mutations in genes that predispose development of cancer.
It is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.
It can compare healthy and mutated DNA sequences can diagnose diseases including various cancers,and can characterize antibody content and can guide patient treatment.
Following the development of fluorescence-based sequencing methods with a DNA sequencer,[6] DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions, such as clusters of genes or operons, full chromosomes, or entire genomes.
It is the most efficient way to indirectly sequence RNA or proteins.
It is used to study genomes and the proteins they encode.
It is used to identify changes in genes, associations with diseases and their phenotypes.
It is used to identify potential drug targets.
DNA sequencing may be used along with DNA profiling methods for forensic identification, and paternity testing.
The DNA patterns in fingerprint, saliva, hair follicles, ca separate each living organism from another, and detect specific genomes in a DNA strand to produce a unique and individualized pattern.
The structure of DNA has four bases: thymine (T), adenine (A), cytosine (C), and guanine (G).
DNA sequencing refers to then determination of the physical order of these bases in a molecule of DNA.
DNA is composed of two strands of nucleotides coiled around each other, linked together by hydrogen bonds and running in opposite directions.
Each strand is composed of four complementary nucleotides adenine (A), cytosine (C), guanine (G) and thymine (T) with an A on one strand always paired with T on the other, and C always paired with G.
Each strand to be used to reconstruct the other, an idea central to the passing on of hereditary information between generations.
Next-generation or second-generation sequencing (NGS) methods, distinguish them from the earlier methods.
NGS technology is typically characterized by being highly scalable, allowing the entire genome to be sequenced at once.
Place on sites DNA sequencing NGS
Oncogeniicity arises from an aggregate excess of cancer promoting over cancer suppressing operations over time: driving mutations tend to accumulate gradually overtime, with the cancer requiring decades to acquire the full complement of cooperating events.
Cancer genes are affected by driver point mutations, gene fusions and simple chromosome rearrangements