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Table of Contents
Year : 2022  |  Volume : 2  |  Issue : 2  |  Page : 58-66

Liquid biopsy: Decoding cancer in blood

Centre for Biorepository and Biobanking, Health Care Global Cancer Centre, Bengaluru, Karnataka, India

Date of Submission17-Oct-2022
Date of Decision12-Dec-2022
Date of Acceptance23-Dec-2022
Date of Web Publication06-Feb-2023

Correspondence Address:
Dr. Abhishek Mohanty
HCG Centre for Biorepository and Biobanking, Health Care Global Cancer Centre, HCG Towers, #8, P. Kalinga Rao Road, Sampangi Ram Nagar, Bengaluru - 560 027, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_17_22

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The molecular landscape of the tumors has been typically established using the surgical or biopsy tissue samples resulting in a sampling bias offering only a single snapshot of tumor heterogeneity from the tissue-based tumor profiles. A rapid understanding of such a bias over the years has helped in procuring a precise portrait of the tumors. This practice has positioned the employability of currently employed molecular analysis of the circulating markers in blood and several other body fluids, such as urine, saliva, and pleural effusions, using liquid biopsies. The genomic profiling of the circulating markers such as circulating circulating tumor DNA (ctDNA), circulating tumor cells, or even RNA, proteins, and lipids as part of exosomes has not only guided the monitoring of response to treatment but also the drug resistance and minimal residual disease. The tumor educated platelets (TEPs) and their biological mechanisms driving the influencing of platelets by tumor cells are beginning to unearth TEPS as dynamically predominant components of liquid biopsy. Here, the biology, methodology, and clinical applications of liquid biopsy biomarkers are highlighted. The article puts forth how technological advances have catapulted cancer diagnosis via liquid biopsy in the last decade to obtain a tumor-derived genetic information for its exploitation toward personalized patient care so that liquid biopsy can come into routine clinical practice.

Keywords: Blood, cancer diagnostics, carcinomas, circulating tumor cells, circulating tumor DNA, liquid biopsy, noninvasive

How to cite this article:
Mohanty A. Liquid biopsy: Decoding cancer in blood. J Precis Oncol 2022;2:58-66

How to cite this URL:
Mohanty A. Liquid biopsy: Decoding cancer in blood. J Precis Oncol [serial online] 2022 [cited 2023 Jun 8];2:58-66. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/2/58/369211

  Introduction Top

̶Cancer” known as the invincible disease has superseded cardiovascular diseases as the most influential factors impacting mortality in developed nations scale to be ranked as the topmost killer disease of the world. The “holy grail” of never-ending global cancer cure remains a far-reaching possibility making cancer the most dreadful disease of all times.[1] In the year 2018 out of the 9.6 million cancer deaths around the world, the USA alone documented >1,735,350 diagnoses of cancer last year amounting to more than 609,640 deaths.[2],[3] The long-standing fight against the cancer has stimulated the need for a persistent effort toward more penetrative but less invasive cancer screening programs for its early detection. The chances of recuperation for a cancer patient as well as administrating personalized treatment regimens can be accelerated with earlier diagnosis aiding in routine monitoring of the tumor progression.

The concept of the diagnostic biomarkers by grabbing these molecules as a tumor byproduct has used the most fundamental properties of cancer progression and dissemination to distant sites showing its aggressiveness. The pursuit to understand the progression of cancer has led to identify molecular markers thus released into circulation called the “tumor circulome.” The basic idea has simplified diagnostic procedures and has led to the development of better diagnostic tests as well as more efficient ways to detect these biomolecules in circulation.[2],[3] This has replaced traditional invasive methods like biopsy and mutation detection with noninvasive approaches. This paper provides an overview of the recent advances in liquid biopsies for detecting tumor-derived genetic material, and discusses the tumor circulome, which is composed of circulating tumor DNA, circulating tumor cells, extracellular vesicles, and circulating tumor RNA, all of which have not yet been fully characterized. Recent technical advancements have had a deep diving influence on redefining the therapeutic value of liquid biopsies, and this article explores the implications of this shift toward non-invasive customised patient care. The noninvasive based individualized patient care approach boosted by the recent technological advances in the field with its deep-diving effects in redefining the clinical value of liquid biopsies is discussed in detail.

  Conventional Practices For Diagnosis of Cancer Top

Customarily, the routine procedures engaging methods for tumor biopsy going through the surgeon's scalpel and invasive protocols have continued to be the preferred choice of standard of care for diagnosis of solid tumors. Currently, the existing approach for cancer diagnosis is mostly invasive involving the examination of tumor tissue through either removing cells using a small needle (fine-needle aspiration cytology) or histological examination of a biopsy or surgical excision specimen. Lately, the analysis of solid tumors obtained from surgical specimens and the information therein concerning genetic alterations in tumors is exploited for diagnostic, prognostic, and treatment purposes. Notwithstanding such progress in the field of molecular pathology and cancer diagnostics, sample preparation modules for such analysis still count on the invasive excision of tumors or a fine-needle aspiration for pathological examinations. However, owing to the invasiveness of these methods being followed for diagnosis, they do come with limitations and clinical complications including the restricted sample size procured from fine-needle aspirates or core-needle biopsies amounting to compromised tissue samples for molecular analysis which is not a limited issue in case of surgically resected specimens. Furthermore, tissue biopsy reflects only a spatial and a single snap-shot information of the malignant tumor, thereby missing out in representing the true nature of tumor heterogeneity, i.e., a landmark of most advanced cancers. Tumors, specifically solitary tumors, also exhibit intra-tumoral (same tumor showing different genetic profiles) as well as inter-metastatic heterogeneity (heterogeneity exists between metastases within the same patient) which will be missed out upon a solid tumor biopsy or resection of only a part/section of a tissue. Majority of surgically resected tumors that are processed for molecular analysis are fixed in formalin and embedded in paraffin blocks (Formalin-Fixed Paraffin-Embedded Blocks, FFPE). As a result of this treatment, permanent mutations, crosslinking, fragmentation, and denaturation of DNA and RNA are introduced, rendering them completely unsuitable for molecular testing or cancer sequencing. In this situation, it is possible to take multiple biopsies of the patient's primary tumour and metastases; however, the complexity of obtaining the tissue sample while considering the patient's comfort, finances, and risks, as well as surgical complications such as inaccessible tumours that can seed/invade adjacent sites, render the multiple biopsy options ineffective. Due to these constraints, putative therapeutic biomarkers cannot be detected early enough during treatment to prevent therapeutic resistance from developing. To be able to use precision oncomedicine and therapy, we need relevant information regarding the tumor genetic and molecular profile.

  Capturing Tumor DNA/Cells in Circulation With Liquid Biopsies Top

The need of the hour is to create fast but less expensive, affordable, and painless approaches which could monitor the tumor-specific genetic alterations, possibly the biomarkers expressed in tumors upon exposure to therapy. Single/tissue biopsies aren't the gold standard for a variety of reasons, including patient risk, procedural cost, sample preparation accuracy, and incompatibility with longitudinal clinical surveillance. Novel procedures have been developed to circumvent the limitations of invasive testing and to monitor mutations and other changes in tumour genetics and tumour dynamics without the need for repeated biopsies. Mandel and Metais in 1948 for the first time found the presence of circulating free DNA (cfDNA) and RNA in human blood with no knowhow of their chemical and clinical relevance at that time.[4] Coincidentally, Dennis Lo,[5] a chemical pathologist, reported the presence of Y chromosomes from the fetus in mother blood carrying male babies. This was probably the first instance of circulating DNA and its utility in prenatal diagnosis paving the way forward toward the identification of fetal changes like germline point mutations, aneuploidy, and genetic aberrations such as Down's syndrome.[5]

Astoundingly, scientists reported in 1977 that the levels of cfDNA were higher in blood of the diseased notable cancer patients than healthy individuals, indicating that it is possible to screen for the presence of cancer through a simple blood test.[6] Adding on, 17 years later, it became clear that the cfDNA indeed had mutations that represented the hallmarks of cancer, an evidence by itself that these mutations originated from the tumors.[7],[8] Hence, the term “circulating tumor DNA (ctDNA)” in molecular oncology came into prominence. The appearance of ctDNA/fragmented genomic material in circulation, floating spontaneously in the bloodstream as a shed out of the ruptured material from cancer cells post apoptosis and death by necrosis, has recently been widely documented and investigated by molecular pathologists. The smaller fragment size of cfDNA (~166 bp) and ctDNA (~130–150 bp) advocates the release of DNA to the blood via apoptosis in addition to necrosis.[9],[10],[11],[12],[13],[14] It has been seen that tumor vascularity, size, and locale determine the passive shedding of the cfDNA into the blood from cells undergoing apoptosis and necrosis.[15] Some of the parallel mechanisms for the release of cfDNA like lysis of CTCs and active tumorigenic secretions are under investigation.[16] Following these events, debris removal molecules like infiltrating macrophages generally mop up the cellular waste from the normal cells, but the same becomes an arduous task for rapidly multiplying, large-sized tumor cells where these scavenging cells do not deal with the time scale of cancer cell division and multiplication to do their assigned job. Thus, the occurrence of the floating cancer cells facilitated by their detachment from primary tumor mass into the bloodstream gave rise to what is nowadays popularly known as CTCs, and predominantly, owing to their prevalence in the blood of patients with solid tumors, including breast, prostate, lung, and colon, the quantification of CTC has emerged as a marker for tumor growth as well as for defining tumor aggressiveness.

The CTCs along with their DNA, being the true repertoire of the tumor genetic characteristics, can truly render the precise genetic and molecular information as an edge over the tissue biopsy. Burgeoning knowledge in the field of tumor genomics, enhancements in targeted therapies, and escalating improvisations in the DNA-sequencing technologies have catapulted the physicians' and oncologists' inclination in utilizing ctDNA as an easy target for noninvasive cancer diagnostic tool. Furthermore, the measurement of tumour DNA (ctDNA) in the bloodstream and the identification of CTCs in the bloodstream are now very much achievable, allowing scientists to study the blood in the form of “Liquid Biopsies.” Eventually, CTC and ctDNA enumerations in blood samples would disclose treatment success, information regarding minimal residual disease, and acquired tumour resistance, assisting as a tool to obtain an enriched picture of the patient's malignancy, leading to accurate timedependent patient monitoring. For instance, the shorter half-life of the ctDNA, i.e., about < 2 h, contributes to a better and unambiguous of tumors' instant status real time rather than its past changes compared to protein biomarkers such as prostate-specific antigen for prostate cancer[17] or cancer antigen (CA) 15-3 for breast cancer recurrence[18] that float in the blood for weeks. Additionally, a couple of striking evidence have revealed the presence of ctDNA to be more sensitive than protein biomarkers for the detection of breast and bowel cancers.[19],[20] Undeniably, the detection of protein markers has been the gold standard method for minimally invasive diagnosis, screening, and postoperative follow-up in cancer management, however, the high falsepositive rates may be due to their elevated levels as a result of physiological perturbations, which can impede the treatment process. Justifiably, the emergence of technological advances and measures for detection of ctDNA which surfaced up with its discovery has taken over the tumor dynamics marker with clear edge over the conventional protein biomarkers used over the years. The information gathered from the CTCs or the genetic/molecular analysis of ctDNA about tumor-associated genomic aberrations is nowadays spearheading the designing of the guidelines for precision oncology such as prognostic evaluation, patient stratification toward target therapy, treatment efficacy, and mechanisms of refractory cancers. As of late, the only components of clinical testing approved by the US Food and Drug Administration (FDA) are the ctDNA and CTCs, which are currently being used to target the tumor genome. However, the other target components of tumor circulome and promising constituents of liquid biopsies under intense research and holding equal potential as ctDNA and CTCs for molecular test development are the EVs, circulating tumor RNA (ctRNA), and TEPs.[2]

One of the newest components in liquid biopsy, tumoreducated blood platelets (TEPs) are among the next and promising prospects of liquid biopsy. Platelets, the essential component of the tumor microenvironment (TME), have been known in tumor biology to govern tumorigenic events such as carcinogenesis, tumor growth, tumor angiogenesis, tumor-related inflammation, and tumor metastasis. Increasing literature precedence displays a tumor cell-circulating platelet interaction being implicated in these events helping in tumor dissemination events[21] [Figure 1]. Furthermore, it has been seen that the fluctuating platelet numbers and subsequent platelet–lymphocyte ratio have been used to be detrimental in prediction of cancer prognosis, cancer prevention, chemotherapy development, and survival prolongation over the few decades.[22],[23]
Figure 1: Tumor cell-circulating platelet interaction: Tumor microenvironment facilitates interplay between tumor cells and circulating platelets leading to the educating the platelets or forming the tumor-educated platelets or TEPs, thereby influencing the regulation of tumor growth, tumor metastasis, dissemination, and angiogenesis. The circulating platelets provide and orchestrate the formation of a cloak containing aggregated platelets encircling tumor cells, sheltering the tumor cells from immune defense, subsequently augmenting tumor dissemination and metastasis

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Two of the pioneering observations in the early 19th century laid the foundations for bringing forth the concept of TEPs. One was the finding by Trousseau in 1868 observing for the first time the occurrence of spontaneous coagulation as a commonality in cancerous patients suggestive of the implications of the circulating platelets in development of cancer.[24] Moreover, the other was by Billroth in 1877 explaining the presence of the multiple blood clots filled with specific tumor elements as integral players in tumor metastasis, thus pinpointing toward a direct tumor cell-platelet interaction being involved in tumorigenesis.[25],[26],[27] Subsequently, the interplay between megakaryocytes, platelets, and cancer that seems complex has been unfolded due to the advent of sensitive molecular techniques, thereby helping to discover the tumor-educated platelets (TEPs).[28],[29],[30],[31],[32] The education of platelets, the enucleated cells, and the second most circulating cell types by the tumor cells happens with the transferring of the associated biomolecules, the RNAs. Therefore, the TEPs as the name suggests act as the best source of noninvasive biomarker in the form of RNA panels constituted by certain RNA signatures from cancer patients.[29] Likewise, the TEPs have a significant clinical weightage in understanding tumor aggression and progression due to ease of identifying their location and their molecular traits from the spliced TEP RNA surrogate signatures.

The mechanistic insights and the extracellular milieu affecting the TEP RNA trove are under constant investigation till date. However, present-day literature and patient data rely on the TME-based external factors which regulate the intraplatelet premature mRNA (pre-mRNA) splicing. Cancerous patients exhibit morphologically an activated state of platelets marked by the expression of membrane markers like p-selectin that is accompanied by the accelerated occurrence of thromboembolism in such populations. The platelets mostly enucleated in nature and shielded by their precursor cells, the megakaryocytes, seen in bone marrow and lungs, and they the sites for their biogenesis maturation and tumor metastases interactions.[33],[34] Furthermore, these tumor interaction sites are involved in the release of cytoplasmic pre-mRNAs which undergo splicing to form mature mRNAs, and then translation into proteins. This may be triggered by external stimuli or might be influenced by the presence of LPS from bacteria and staphylococcal-derived alphatoxin.[35],[36],[37] The exact identification of the queue-restricted splicing factors or biomolecules released from either the tumor cells or the TME consisting of stromal and immune cells defining the splicing events, remains a study for investigation. The platelets as a cellular response to external stimuli release the RNA signaling complexes to other cells via microparticles known as platelet-derived microparticles (PMPs),[38],[39] an event which characterizes tumor progression. PMPs that make up almost for 70%–90% of all EVs justifying their abundant presence in a peripheral blood, take an active part in angiogenesis, metastasis, and multidrug resistance following the activation or maturation of platelets. The second type of vesicles, namely the tumor-derived vesicles, is still able to keep their biomolecules in platelets such as the proteins and nucleic acids, the cfDNA inherent to platelets throughout their maturation process.[29],[40],[41] TEPs contain a pool of active mRNA that is delivered to them after they digest the circulating or spliced mRNA generated in response to external stimuli. These mRNA can serve as markers for TEP-based liquid biopsy. Moreover, platelet protein synthesis and platelet signaling can be a consequence of their interaction with tumor cells directly.

Furthermore, the tumor interaction with platelets can drive the protein synthesis and signaling in platelets contributing to the megakaryocyte-derived proteins, endocytosed proteins, and proteins being translated in individual platelets. Only some anecdotal evidence suggests that platelets undergo cross-talk driven and tumor-influenced RNA alterations[42],[43] that determine how they are educated resulting in intraplatelet splicing. These driving events can be explained by changes in platelet RNA splicing, exon skipping, differential RNA binding protein activity[44],[45] and altered aging/turnover.[29],[45]

Following the education of platelets post their direct tumor interactions, the truly tumor-derived proteins are segregated from the independently sequestered biomolecules such as proteins and RNA as a characteristic part of tumorigenic events. It has been shown that the transcripts directly transferred to platelets are integral to or contribute to the development of cancer-specific TEP RNA biomarkers in glioblastoma,[41],[46] PCA3, FOLH1, KLK2, KLK3 and NPY in prostate cancer,[41],[47] EML4ALK in NSCLC,[48] mutants of KRAS, EGFR, or PIK3CA, or[49] HER2 in NSCLS or breast cancer. Best et al. in 2015 through their path-breaking work defined the way ahead for predictive pan-cancer diagnostics, by being able to sequence and measure differentially spliced RNA profiles in platelets across six cancer types. For this study, the successful extraction of characterized TEPs was done from a cohort of patients (non-small cell lung carcinoma, colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, and breast cancer).[49]

As a result of this work, both the metastasized patients and those with localized tumor could be separated from the normal individuals with an accuracy of 96% and aiding in defining the tumors' anatomical position with 71% accuracy probably pinpointing tumor-specific RNA splicing in platelets. As a follow-up, the same group used particle-swarm optimization-boosted algorithms to platelet RNA-Seq libraries to create an RNA biomarker panel helping in differentiating lung cancer patients with inflammation from the healthy ones.[45]

Undeniably, the blend of molecular profiling using TEPs with the other molecular resources such as EVs, ctDNA, CTCs, and imaging biomarkers gives an added advantage toward developing companion diagnostics in sync with the liquid biopsies. Nevertheless, impounding concerns still worrying the scientists in the field about the TEPs are the dynamic protein and transcriptome changes happening in the TEPs keeping in view of a nuclear trait? Furthermore, in order for a single tumor cell to educate thousands of platelets, and to characterize this physiological milieu, the exact quantification of the tumor cells must be performed before conditions are evident for platelets to be educated. Thirdly, no complete studies have reported the signatory total miRNA profile of the TEPs till date. Consequently, to supplement long read sequencing and TEP transcriptome mapping, a fill in the gap will be the addition of proteomics of TEPs to analyze the platelet proteome. This will enable the development of simple detective methods for investigating epigenetic and epitranscriptural features.

  Circulating Tumor DNA and Technical Advancements Meeting the Needs of Liquid Biopsy Top

It is important to note that the detection and analysis of ctDNA come with a number of technical constraints relating to segregating ctDNA from normal cfDNA and the sensitivity of detection. Determining the limit of detection levels for lower levels of ctDNA will affect the frequency of tumor variants, particularly those found in early-stage cancers. The difficulty in distinguishing between ctDNA and normal cell-free DNA is compounded by the prevalence of single basepair substitutions among the somatic mutations found in cancer genomes, including gene rearrangements (EGFR and KRAS) rearrangements (EML4ALK), gene amplifications (HER2 and MET), and translocations. These kinds of genetic alterations are remarkable for the tumor cell genome or precancer genome well supported by their absence in healthy individuals providing a reassurance of the efficacy of ctDNA as an invaluable biomarker. Due to the low input of ctDNA in total cfDNA of often 0.01,[14] traditional DNA analysis techniques (such as Sanger sequencing) have compromised their sensitivity. As a result of advances in technology, these defects in molecular diagnostics have been overcome with improved detection techniques, which have demonstrated superior accuracy and precision. This has transformed the process of molecular and analytical techniques driving ctDNA detections, and a paradigm shift is seen toward polymerase chain reaction (PCR) or next-generation sequencing (NGS) based reporting of ctDNA mutations. The most promising of these new techniques are the allele-specific polymerase chain reaction (PCR)[7] and the modified quantitative PCR (qPCR) method that has been approved by the FDA for the cobas EGFR mutation test.[50],[51],[52],[53] For the analysis of ctDNA, a number of technologies have developed with high analytical sensitivity and specificity. The methods include digital PCR (dPCR), droplet dPCR,[51] Beads, Emulsions, Amplification, and Magnetics (BEAMing), pyrophosphorolysisactivated polymerization, taggedamplicon deep sequencing (TAMSeq), parallel sequencing, and qPCR.[53] Astonishingly, Bert Vogelstein and Kenneth Kinzler developed the technique called BEAMing at Johns Hopkins which traps the DNA onto the magnetic beads. The beads, after isolation and counting, can detect ctDNA to a limit of 10,000–1 in contrast with healthy c/normal cell DNA. In addition to their cost-effectiveness and superior sensitivity, PCR-based assays suffer from low multiplexing capacity, thus limiting the number of loci that can be analyzed simultaneously to a limited number.[54] Additionally, the genomewide approach using nontargeted sequencing enables identifying and prioritizing tumor-specific alterations in the absence of possible aberrations that may be present. NGS of ctDNA sequences intends toward cataloging and resourcing the information to gather the collated genomic information across various cancers and subtypes followed by molecular stratification of tumors.

The correlation of the NGS data can be utilized for standardization of patient treatment and monitor prognosis besides their proven role in detecting point mutations such as insertions, deletions or rearrangements, and copy number variations and gene fusions. Such molecular events are known to modulate both tumorigenesis and resistance mechanisms emerging from preexisting clones' posttreatment. Thus, the coming years look forward toward mixing the dPCR and NGS complementing each other to drive liquid biopsy and redefine minimally invasive molecular and precision onco-diagnostics. The drug trials battling to develop newer drugs targeting the novel and rare actionable mutations being identified have opened avenues for the urgent need of the molecular testing of an increasing number of mutations per biopsy placing NGS as a better approach for molecular diagnostics than preexisting ones. The forthcoming era of liquid biopsy sailing through NGS hopes to see the evaluation of tumor mutational burden (TMB) as a potential biomarker for NSCLC in response to chemotherapy and immunotherapy. TMB previously was calculated from the whole-exome sequencing (WES) data but of late recent evidence have claimed that TMB estimated from targeted NGS and/or WES of ctDNA has shown to be efficient in predicting response to therapy.[55],[56]

Presently, tumor genotyping procedures are now gearing up and with ctDNA-based molecular analysis taking center stage supported by CTC isolation and enrichment primarily on blood (plasma and serum) samples.However, recent literature have put forth other body fluids such as urine,[57] cerebrospinal fluid,[19],[58],[59] saliva,[60],[61] in lung cancer-the bronchial washings and pleural fluids[62],[63] as preferred alternatives PCR/NGS based molecular analysis of ctDNA. This has definitely raised the bars for addressing the sensitivity and specificity challenges in sample paucity-affected cancer diagnostics. Put together, in other words, “Liquid Biopsy is like barcoding the cancer in the blood,” and via this, researchers and medical oncologists around the globe are finding out things about individuals' cancers that astonish them.

  “Liquid Biopsy,” A Game Changer in Cancer Diagnosis and Treatment Top

Liquid biopsy with its credible penetrating consequences on genetic testing, diagnostics, and patient monitoring both pre- and posttherapy has catapulted the field of clinical cancer diagnostic and precision oncology. The development of this noninvasive technique based on liquid biopsy has been possible as a result of several of these advantages and technical applications. As well as allowing early and timely disease detection, liquid biopsy has the potential to provide an accurate assessment of metastasis in real time. This is done by capturing and analyzing biomarkers that are similar to tissue. By using fresh DNA, researchers can investigate primary tumors and metastases through simple, noninvasive approaches without the inconvenience of sample degradation hampered by preservatives. Periodic and dynamic monitoring of tumor dynamics can further enhance the efficiency of tumor heterogeneity assessment. This is done in order to observe molecular changes occurring within the tumor, which are delayed in solid biopsies as a result of time, cost, and logistical constraints. An analysis of this type can be helpful in determining whether a tumor has entered dormancy. Liquid biopsy is expected to provide a more integral portrait of molecular events underlying tumor progression as compared to a single tissue-take.[64],[65],[66] In the year 2016, with the FDA approval of the cobas EGFR Mutation Test v2, Roche Diagnostics, a milestone was achieved in the field of companion diagnostics, this being the first approved ctDNA-based test for lung cancer.[50] This test presently has a therapeutic use and plays a vital role in patient treatment an intervention utilizing EGFR-tyrosine kinase inhibitors on the basis of specific EGFR-sensitizing mutations in patients with NSCLC. The first report of combinatorial diagnostic engaging protein and ctDNA as dual biomarkers is now looked ahead as among the first steps in next-generation innovation strategy in early detection and initial assessment of disease. For instance, the test, named CancerSEEK, engaged PCR to concomitantly detect mutations in 16 oncogenes across the cancer panel in combination with quantification of 8 proteins CA19-9, CEA, HGF, OPN, CA-125, myeloperoxidase, prolactin, and tissue inhibitor of metalloproteinase 1. This test with an ability to analyze multiple types of markers blends the multiplexed PCR-based ctDNA mutation detection at 1933 loci with the measurements of validated protein biomarkers and has been a breakthrough approval from FDA for pancreatic and ovarian cancers.[67] The test to determine whether CancerSEEK can detect other cancers, making it a pancancer test, is ongoing and will be completed in the near future.

  Roads to Conquer in Liquid Biopsy Guided Molecular Profiling of Tumors Top

The advent of liquid biopsy has refurbished the face of molecular oncology owing to its bourgeoning and increasing applications developing incredibly over the last 5 years. Nevertheless, in spite of the growing huge and attractive potential of liquid biopsies in early cancer detection for diagnosis to molecular characterization of the tumor for treatment optimization and monitoring, there remains a large set of limitations to overcome and open issues still needing straightforward and unambiguous answers. Does ctDNA provide a truly symbolic portrait of every cancer? Do metastatic tumors release as much DNA as the original tumors? Is the amount of ctDNA released by tumor cells post death the same and comparable with ctDNA extracted in life? Does an accurate picture of tumor burden, or a real-time look at emerging mutations, will actually save patients or improve their quality of life? The standardization of the blood collection protocol to that of ctDNA isolation ensuring less preanalytical variability along with more well-defined quantification methods preceded by broader ctDNA sequencing platforms aiding in discovery of actionable therapeutic targets can facilitate the detection of rare molecular alterations and hot spot mutations to anticipate drug resistance. It is possible to obtain a clear understanding of tumor characteristics, including aggressiveness and overall molecular landscape, by using liquid biopsy approaches in patient screening. In light of this, blood-based liquid biopsy is already being utilized as a benchmark test for confirming sufficient amounts and quality of ctDNA. Adding on, a well-known fact in the preponderance of tumor-derived DNA concentration is the abundance of such a biomarker in proximal body fluid compared with blood at earlier stages of cancer.[68] Consequently, the alternative nonbloodbased body fluid-based liquid biopsy technique is to enhance ctDNA detection rates. By capturing ctDNA in localized tumor cells containing rare mutations, close to the tumor's presumed site, this can overcome the challenges associated with sensitivity and specificity in the molecular technique driven precision oncology era.

  Concluding Remarks Top

In conclusion, the next decade of liquid biopsy-driven blood-based genomic profiling will rule the field of minimally invasive molecular diagnostics. Liquid biopsy will probably provide a handle to create a simple diagnostic power, before we answer certain challenges like will liquid biopsy-driven treatments translate into improved outcomes? So on and so forth, the discovery of TEPs and its biology aiding in developing the TEP-RNA-based liquid biopsy has added a new dimension in molecular oncology that will be there in days ahead, transforming early detection in pan-cancer diagnostics to combinatorial diagnostics toward patient monitoring, and therapy-based cancer diagnostics.

Nevertheless, it would be befitting to say that liquid biopsy remains a simple blood draw that could tell the doctors what they need to know to treat cancer more effectively. However, there still remain many puzzles that need to be cracked before “liquid biopsies” join the ranks of routine and standard medical tests. Hence, irrefutably liquid biopsy is the modern era's best and most promising solution to the increasing complications in modern-day oncology primarily based on biomarker discovery and molecular and genomic testing.

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