Circulating Tumor Cells And Circulating Tumor Dna PdfBy Gayane P. In and pdf 28.01.2021 at 12:38 3 min read
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- Impact of Circulating Tumor DNA (ctDNA) in Patients with Gastrointestinal Malignancies
- Circulating tumor DNA
- Circulating tumor DNA
Despite many advances in the diagnosis and treatment of colorectal cancer CRC , its incidence and mortality rates continue to make an impact worldwide and in some countries rates are mounting.
A biopsy sample of tissue often is tested for specific genetic variations also referred to as mutations that may have a targeted inhibitor that represents a clear optimal treatment for that cancer. However, it may not be possible to get enough tissue from the tumor to do these studies. DNA from our cells, including cancer cells, breaks down as part of its normal life cycle. Testing of ctDNA offers several advantages over attempts at repeated tissue biopsies.
Impact of Circulating Tumor DNA (ctDNA) in Patients with Gastrointestinal Malignancies
Hepatocellular carcinoma HCC and pancreatic cancer PC belong to the most lethal malignancies worldwide. Despite advances in surgical techniques and perioperative multidisciplinary management, the prognosis of both carcinoma entities remains poor mainly because of rapid tumor progression and early dissemination with diagnosis in advanced tumor stages with poor sensitivity to current therapy regimens.
Both highly heterogeneous visceral carcinomas exhibit unique somatic alterations, but share common driver genes and mutations as well. Recently, circulating tumor DNA ctDNA could be identified as a liquid biopsy tool with huge potential as non-invasive biomarker in early diagnosis and prognosis. CtDNA released from necrotic or apoptotic cells of primary tumors, metastasis, and circulating tumor cells can reveal genetic and epigenetic alterations with tumor-specific and individual mutation and methylation profiles.
Hepatocellular carcinoma HCC and pancreatic cancer PC represent two of the most challenging visceral malignancies in oncology with rising incidence and lack of reliable biomarkers for early diagnosis, prognosis, and therapy response.
PC and HCC are estimated to become the second and third respective leading causes of cancer-related death in western countries by Rahib et al. As both carcinoma entities share some common risk factors, either environmental or genetic, combined analyses may provide useful information.
Hepatocellular carcinogenesis is a multistep process occurring in one-third of patients with liver cirrhosis, on the background of chronic infection with hepatitis B or C virus HBV, HCV , alcoholic or non-alcoholic steatohepatitis, and obesity Villanueva PC occurs with increased frequency among individuals with tobacco smoking, type 2 diabetes, obesity, chronic pancreatitis or hereditary risk factors Ryan et al.
Therefore, early diagnosis at surgically manageable stages and early recurrence detection would have a tremendous impact on survival of patients with HCC or PC. In addition, imaging techniques failed to detect early lesions or to distinguish between benign and malignant lesions, so far. In addition to the risk of neoplastic needle tract seeding, minimal invasive solid biopsies by endoscopic-ultrasound-guided fine needle aspiration in PC or percutaneous needle biopsy in HCC cannot accurately track dynamic changes due to high tumor heterogeneity Stigliano and Burroughs ; Yoshida et al.
Cytotoxic chemotherapy agents continue to form the backbone for the treatment of advanced PC limited to the pyrimidine antimetabolites gemcitabine and 5-fluorouracil 5-FU , Topoisomerase I inhibition by irinotecan, the DNA crosslinking agents oxaliplatin and cisplatin, and the tubulin inhibitor paclitaxel Burris et al.
However, the median survival remains 6—11 months. Since , gemcitabine-based combination chemotherapies with the selective epidermal growth factor receptor EGFR tyrosine kinase inhibitor erlotinib could improve the overall survival in locally advanced, unresectable, or metastatic PC Moore et al. However, better therapy response with extended survival time of more than 2 years in small subgroups of patients with advanced PC seems to be connected with exceptionally favorable prognostic factors and molecular characteristics Collisson et al.
Currently, two first-line alternatives to Sorafenib are approved. Therefore, the most effective treatment of heterogeneous cancers like HCC and PC might be a tailored combination of drugs targeting specific genomic and epigenomic alterations.
Routine molecular testing is still performed in clinical diagnostic for targeted therapy and prognostic stratification in cancer entities, such as breast cancer, melanoma and leukemia El-Deiry et al. In addition to environmental factors, recent genome-wide association studies revealed that genetic and epigenetic abnormalities might be significant determinants of HCC or PC susceptibility with important influence on the individual predisposition to disease progression, e.
Moreover, several recent studies have shown that identification of molecular biomarkers and real-time monitoring of disease and therapy efficacy in PC and HCC could be achieved by liquid biopsies Tables 1 and 2.
In this review, we discuss recent studies focusing on detection and clinical impact of circulating DNA mutation and methylation as potential biomarker for early diagnosis, prognosis, and therapy response in HCC and PC. Cell-free cf DNA originates from normal cells exported by exosomes as well as from apoptotic and necrotic cells with highly fragmented, double-stranded DNA of approximately — base pair fragments in size being released into the bloodstream. In , Mandel and Metais first reported the presence of cfDNA in human circulation followed by detection in urine, saliva, and other body fluids Botezatu et al.
A recent study showed that most of cfDNA derive from bone-marrow and liver in healthy individuals Sun et al. Examples for clinical applications of cfDNA are the non-invasive prenatal testing for chromosomal aneuploidies by fetal cfDNA in the plasma of pregnant women or the monitoring of graft rejection following organ transplantation by donor-derived cfDNA in the plasma of the recipients Chiu et al.
In , Leon et al. It was postulated that cancer patients have higher levels of cfDNA than healthy individuals Shapiro et al. However, cfDNA levels have also been linked to outcomes in patients with a variety of other physiological and pathological conditions, including exercise, inflammation, circadian rhythm, exposure to smoking, sepsis, and trauma Aucamp et al.
In fact, cfDNA is composed of both coding and non-coding genomic DNA that can be used to examine mutations and polymorphisms, microsatellite instability, and epigenetic methylation Bruhn et al.
Epigenetic changes are considered as an early event in carcinogenesis and might, therefore, be a suitable early diagnostic tumor marker.
Since the extraction of plasma DNA with the same genetic changes as the primary tumor in and identification of mutated KRAS sequences in the plasma or serum from patients with PC in , circulating tumor ct DNA is becoming a research hotspot with high potential as liquid biopsy marker in cancer medicine Sorenson et al.
CtDNA is considered to be released from an increasing proportion of necrotic and apoptotic cells in primary tumors, secondary deposits and circulating tumor cells, corresponding to an increase in ctDNA. CtDNA can be detected by tumor-specific mutants and are less impacted by intratumor heterogeneity than a single specimen of tumor tissue Bettegowda et al.
It is supposed, that ctDNA harbor the same epi- genetic alterations as the originating primary tumor cells. Recently, Heitzer et al. In addition, previous studies have indicated a positive correlation between ctDNA levels and tumor burden in various cancer types with increasing copy numbers of ctDNA per mL plasma in advanced and metastatic tumors, as well as different methylation status of ctDNA to normal cfDNA and blood leukocytes Bettegowda et al. Furthermore, it is hypothesized that ctDNA could be a signaling trigger for cancer progression by horizontal DNA transfer affecting the biology of host cells Bergsmedh et al.
Thus, ctDNA levels may serve as an early biomarker for diagnostic and therapy monitoring, prior to clinically or radiographically measureable changes of tumor burden in patients. Deoxyribonuclease activity and the release of cfDNA by normal cells in peripheral circulation reduces ctDNA concentrations. Therefore, the detection of ctDNA is challenging and necessitates the standardization of extraction procedures to be able to distinguish ctDNA from the large amount of cfDNA.
In the last 2 decades, growing knowledge on non-coding genome functionality and genome-wide sequence variation has improved personalized medicine and molecular oncology. With technological advances broadly categorized into PCR-based and genomic sequencing-based techniques, sensitivity and specificity of biomolecular techniques for ctDNA detection have been highly improved.
Currently, high-throughput next-generation sequencing NGS and digital droplet PCR ddPCR are the most promising methods for the detection of mutations in liquid biopsies. In general, plasma samples are used in preference over serum because of lower concentrations of wild-type DNA Jung et al.
NGS techniques allow the simultaneous assessment and detection of multiple genetic aberrations and copy number changes, including targeted techniques such as enhanced tagged amplicon deep sequencing, or whole-exome sequencing. The aim of NGS is to generate extensive information about the mutation landscape, then to screen the genome and discover new genomic aberrations, e. The whole-genome and whole-exome sequencing could provide more comprehensive information regarding the mutant status of ctDNA.
However, the targeted region deep sequencing, which focuses on a fewer gene loci with ultra-deep sequencing, has gained popularity, because the sequencing region can be customized according to cancer types, sequencing purpose, costs, and turnaround time Leary et al.
Compared to quantitative real-time qRT PCR, samples are processed with a water—oil emulsion to allow for individual droplets to be assessed as a discrete PCR sample and does not rely on external calibrant. Digital PCR has a higher tolerance for enzyme-inhibiting substances thereby improving sensitivity and specificity of mutant DNA detection Hindson et al. In combination with circulating nucleic acids, ddPCR has gained wide applicability in liquid biopsy diagnostic of cancer, especially in the assessment of methylation status to identify epigenetic dysregulation during carcinogenesis and measurement of changes in gene expression to early diagnosis of cancer, the absolute quantification of copy number variations to predict disease progression, or the detection of rare mutations within ctDNA to guide targeted therapy Huggett et al.
NGS profiling of surgically resected HCC revealed a highly heterogeneous cancer caused by the accumulation of genomic and epigenomic alterations Ozen et al.
This comprehensive and integrative characterization of molecular profiling in HCC tissues followed by the identification and quantification of corresponding ctDNA in the plasma may provide powerful data for targeted therapies and monitoring of therapy response. TERT promotor mutations were found to be the most common point mutations in several carcinoma entities with reactivation of telomerase enabling limitless cell proliferation driven by oncogenes Bell et al. DdPCR by Huang et al.
Mainly missense mutations in the DNA-binding domain of TP53 are generally thought to abrogate the tumor suppressor function of p53 as the guardian of the genome.
Loss of p53 function with consecutive dysregulation of apoptosis, cell cycle arrest, DNA repair and metabolic regulation is a prerequisite for tumor initiation and progression in a multitude of human cancers.
However, mutant p53 not only lose tumor suppressive functions of wild-type p53 but also gain new oncogenic properties promoting tumor cell proliferation, angiogenesis, and metastasis.
The mechanism for the accumulation of mutant p53 and its mutational gain of function in tumors is not yet well understood. Liao et al. Similarly, postoperative detection of ctDNA related mutations by An et al. Besides genetic alterations with change of DNA sequence, epigenetic silencing of tumor suppressor genes by promotor hypermethylation has been proven to be present in precursor lesions of HCC.
Aberrant DNA hypermethylation consists of the addition of a methyl residue on cytosines preceding guanosines leading to a condensed chromatin structure without transcriptional activity. Wong et al. High incidence of p16INK4a promoter hypermethylation in ctDNA with significant decrease in postoperative blood samples was shown to be a useful marker in the detection and monitoring of HCC Wong et al.
Correspondingly, Huang et al. Further common tumor suppressing and cell cycle regulation-related genes with promoter hypermethylation are the adenomatous polyposis coli APC on chromosome 5q21 and the Ras association domain family protein 1A RASSF1A genes on chromosome 3p Mohamed et al.
Chan et al. Tangkijvanich et al. Combined detection of ctDNA methylation markers was performed by several studies to improve the efficiency in early HCC diagnostic. Xu et al. Although a multitude of aberrant methylated genes could be identified as prognostic target in HCC, there is no recognized biomarker confirmed in multiple centers Han et al. In the last decade, comprehensive genomic analysis allowed important advances in the understanding of the molecular pathogenesis of PC with reclassification in different specific subtypes Bailey et al.
Several studies using different techniques could reveal that reproducible molecular subgroups with consistent alterations in genes and signaling pathways are emerging in PC. Evaluating specimens of resected PC by a combination of whole-genome sequencing and deep-exome sequencing, Bailey et al.
However, there is still a lack of consensus in the clinical applicability of current PC subtyping approaches. Interestingly, the dynamics of total cfDNA concentration correlated positively with tumor burden following chemotherapy and might be a promising tool for early response prediction and therapy surveillance in patients with advanced PC. Chen et al. Berger et al. TP53 and KRAS mutation levels were significantly decreased during treatment, and on the other hand significantly increased during tumor progression correlating with progression-free survival Berger et al.
However, current biomolecular technologies confirm the genetic heterogeneity of PC with a large number of low-frequently mutated loci and a large fraction of patients who does not harbor mutations in KRAS or TP53 Martinez et al.
Methylation analyses of ctDNA that reveal epigenetic alterations with more or less diagnostic and prognostic impact reflect the remarkable heterogeneity in PC patients. Henriksen et al. Further methylation analyses of ctDNA were able to differentiate PC from chronic pancreatitis and healthy controls Henriksen et al. Confirming study results of Yi et al.
So far, in relatively, few PC patients no single ctDNA promotor hypermethylation with adequate sensitivity and specificity has been found. Furthermore, serial ctDNA studies following the methylation profile of PC patients in accordance with treatment and tumor recurrence are lacking. CtDNA has gained considerable attention as novel liquid biopsy marker for cancer detection in asymptomatic individuals and of residual disease.
Indeed, ctDNA has huge clinical potential for prognostication and response monitoring of patients with HCC and PC characterized by high tumor heterogeneity and dismal prognosis.
However, although literature regarding ctDNA assays and molecular profiling is rapidly growing, its translation into clinical applicability is highly complex. Limited data are available regarding the blood draw procedure and pre-analytical variables that increase degradation of cfDNA or contamination by cellular genomic DNA derived from leukocyte lysis Lee et al. There is consensus that cfDNA analysis requires special processing and handling by using cell-stabilization tubes and avoiding repeated freeze—thaw cycles.
Furthermore, patient-related factors as medical treatment, smoking, exercise, age-related clonal hematopoiesis, inflammation or cardio-pulmonary disorders may contribute to the release of cfDNA.
The proportion of ctDNA as a fraction of cfDNA varies substantially between different patients, and different subclonal variants might be identified. Therefore, allele fractions of variants in ctDNA need to be interpreted with great caution.
In the last decade, numerous platforms for genotyping of cfDNA have been developed.
Circulating tumor DNA
Hepatocellular carcinoma HCC and pancreatic cancer PC belong to the most lethal malignancies worldwide. Despite advances in surgical techniques and perioperative multidisciplinary management, the prognosis of both carcinoma entities remains poor mainly because of rapid tumor progression and early dissemination with diagnosis in advanced tumor stages with poor sensitivity to current therapy regimens. Both highly heterogeneous visceral carcinomas exhibit unique somatic alterations, but share common driver genes and mutations as well. Recently, circulating tumor DNA ctDNA could be identified as a liquid biopsy tool with huge potential as non-invasive biomarker in early diagnosis and prognosis. CtDNA released from necrotic or apoptotic cells of primary tumors, metastasis, and circulating tumor cells can reveal genetic and epigenetic alterations with tumor-specific and individual mutation and methylation profiles.
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PDF | Solid tumors derived from epithelial tissues (carcinomas) are responsible for 90% of all new cancers in Europe, and the main four tumor.
Circulating tumor DNA
Circulating tumor DNA ctDNA testing is rapidly emerging as a new tool for scientists and oncologists treating gastrointestinal cancers. The varied numbers of testing platforms, either research or the ones that are commercially available, are helping us understand several fundamental aspects of tumor The varied numbers of testing platforms, either research or the ones that are commercially available, are helping us understand several fundamental aspects of tumor biology. For example, tumor heterogeneity and tumor evolution is a concept that we as oncologists have always known about and seen in patients being treated for any of the gastrointestinal cancers. However, it is now through ctDNA testing that we are truly gaining an understanding of how things are evolving and what are the mechanisms underlying this change.
As a tumor grows, cells die and are replaced by new ones.
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