Liquid biopsies in cancer

Liquid biopsy,the detection of biomarkers from body fluids,has been widely used on circulating tumor cells(CTCs),circulating tumor DNA(ctD NA),circulating tumor microRNA,exosomes and other kinds of molecular characterization.Cancer,a series of comprehensive mechanisms,presents in different molecular forms that could be used in clinic for diagnosis,treatment,drug monitoring and prognosis judgement via liquid biopsies.Liquid biopsies are available to tumor heterogeneity,resistance,progression and metastases in clinic.Technologies for detection and analysis was improving for the obvious limitation of liquid biopsies,that leads the future directions.In this review,we provided critical views of liquid biopsies on cancer,including different methods of liquid biopsies and applications of liquid biopsies in current clinical trials and practice.

Potential utility of liquid biopsies in the management of patients with biliary tract cancers:A review

Biliary tract cancer,comprising gallbladder cancer,cholangiocarcinoma and ampullary cancer,represents a more uncommon entity outside high-endemic areas,though global incidence is rising.The majority of patients present at a late stage,and 5-year survival remains poor.Advanced stage disease is incurable,and though palliative chemotherapy has been shown to improve survival,further diagnostic and therapeutic options are required in order to improve patient outcomes.Although certain subtypes of biliary tract cancer are relatively rich in targetable mutations,attaining tumour tissue for histological diagnosis and treatment monitoring is challenging due to locoregional anatomical constraints and patient fitness.Liquid biopsies offer a safe and convenient alternative to invasive procedures and have great potential as diagnostic,predictive and prognostic biomarkers.In this review,the current standard of care for patients with biliary tract cancer,future treatment horizons and the possible utility of liquid biopsies within a variety of contexts will be discussed.Circulating tumour DNA,circulating microRNA and circulating tumour cells are discussed with an overview of their potential applications in management of biliary tract cancer.A summary is also provided of currently recruiting clinical trials incorporating liquid biopsies within biliary tract cancer research.

Use of liquid biopsies in gastrointestinal cancers

The use of liquid biopsies is a relatively new tool in diagnosis and management of gastrointestinal cancers and is actively being investigated.Liquid biopsies have become extremely popular in cholangiocarcinoma and pancreatic cancer research.With more prospective trials using this tool for early diagnosis,liquid biopsies may become an important part of cancer management.

Tumor circulome in the liquid biopsies for digestive tract cancer diagnosis and prognosis

Digestive tract cancer is one of the main diseases that endanger human health.At present,the early diagnosis of digestive tract tumors mainly depends on serology,imaging,endoscopy,and so on.Although tissue specimens are the gold standard for cancer diagnosis,with the rapid development of precision medicine in cancer,the demand for dynamic monitoring of tumor molecular characteristics has increased.Liquid biopsy involves the collection of body fluids via noninvasive approaches,and analyzes biological markers such as circulating tumor cells,circulating tumor DNA,circulating cell-free DNA,microRNAs,and exosomes.In recent years,liquid biopsy has become more and more important in the diagnosis and prognosis of cancer in clinical practice due to its convenience,non-invasiveness,high specificity and it overcomes temporal-spatial heterogeneity.Therefore,this review summarizes the current evidence on liquid biopsies in digestive tract cancers in relation to diagnosis and prognosis.

Early detection of non-small cell lung cancer in liquid biopsies by ultrasensitive protease activity analysis

Aim:A significant fraction of mortalities from non-small cell lung cancer could be prevented,if the cancer would be diagnosed earlier.Nanobiosensors for the ultrasensitive detection of active proteases in serum were designed to detect a significant protease activity signature of non-small cell lung cancer(stage I and higher).Methods:We determined the activity of nine protease biomarkers in the sera of non-small cell lung cancer patients and compared them with the protease activities of a control group of healthy human subjects using optical nanobiosensors.They consist of a central Fe/Fe3O4 core/shell nanoparticle with an attached Fluorescence resonance energy transfer-pair[tetrakis-carboxyphenyl porphyrin(TCPP)and cyanine 5.5].TCPP is attached to the central nanoparticle via a protease-cleavable tether,whereas cyanine 5.5 is tethered permanently to the dopamine-layer surrounding the nanoparticle.Results:Based on the activity pattern of urokinase plasminogen activator,matrix metalloproteinases 1,2,3,7,9,and 13,and cathepsins B and L as well,non-small cell lung cancer could be detected at stage I by means of a liquid biopsy.Conclusion:This feasibility study,comprising 33 non-small cell lung cancer patients and 20 apparently healthy subjects,clearly demonstrated the feasibility of minimally invasive early diagnosis of non-small cell lung cancer,starting with stage I.

The power of microRNAs as diagnostic and prognostic biomarkers in liquid biopsies

In the last decades,progresses in medical oncology have ameliorated the treatment of patients and their outcome.However,further improvements are still necessary,in particular for certain types of tumors such as pancreatic,gastric,and lung cancer as well as acute myeloid leukemia where early detection and monitoring of the disease are crucial for final patient outcome.Liquid biopsy represents a great advance in the field because it is less invasive,less time-consuming,and safer compared to classical biopsies and it can be useful to monitor the evolution of the disease as well as the response of patients to therapy.Liquid biopsy allows the detection of circulating tumor cells,nucleic acids,and exosomes not only in blood but also in different biological fluids:urine,saliva,pleural effusions,cerebrospinal fluid,and stool.Among the potential biomarkers detectable in liquid biopsies,microRNAs(miRNAs)are gaining more and more attention,since they are easily detectable,quite stable in biological fluids,and show high sensitivity.Many data demonstrate that miRNAs alone or in combination with other biomarkers could improve the diagnostic and prognostic power for many different tumors.Despite this,standardization of methods,sample preparation,and analysis remain challenging and a huge effort should be made to address these issues before miRNA biomarkers can enter the clinic.This review summarizes the main findings in the field of circulating miRNAs in both solid and hematological tumors.

Magnetic nanotechnologies for early cancer diagnostics with liquid biopsies:a review

Liquid biopsy has become an emerging technology in the detection of cancer related biomarkers as well as the continuous monitoring of cancer treatment.There has been extensive research on the applications of magnetic nanotechnologies in liquid biopsies from the separation of target analytes to the detection of cancer biomarkers.Magnetic separation plays an important role in increasing both the efficiency and sensitivity of the liquid biopsy process.The detection of cancer biomarkers through magnetic nanosensors also possesses many advantages such as low background noise,high sensitivity,short assay time,and the ability to detect multiple biomarkers at the same time.This review focuses on the recent advances of magnetic nanotechnologies in liquid biopsies for cancer detection and its future potential in comparison with other technologies.

The role of cell free DNA and liquid biopsies in haematological conditions

Cell free nucleic acids(CFNAs)are nucleic acids released from cells that circulate within bodily fluids.Recent advances in molecular techniques have led to the ability to interrogate CFNAs in a clinically meaningful way,for example the identification and assessment of foetal CFNAs in maternal blood,allowing minimally invasive testing for foetal genetic abnormalities.The majority of CFNAs arise from haemopoietic cells,making it a particularly rich source of genetic information in haematological conditions.Furthermore,the innate genetic heterogeneity of haematological malignancies,as epitomised by multiple myeloma,lend itself well to“liquid biopsies”.This approach promises to provide a more wholistic assessment of whole disease genetics,especially when contrasted against the current gold-standard of single site tissue biopsies.This review briefly summarises the definitions and physiology of CFNAs,both cell free DNA(cfDNA)and extracellular RNA(exRNA),before exploring the literature surrounding the current and future roles of cfDNA in the haematological malignancies and patient care.

Development of a TCR beta repertoire assay for profiling liquid biopsies from NSCLC donors

Aim:The aim of this study was to demonstrate the utility of T-Cell receptor beta(TCRβ)sequencing as a robust method for assessing T-cell repertoire changes in donors with non-small cell lung cancer(NSCLC).We further demonstrated the use of the assay by monitoring repertoire modulation in a defined model antigen system,cytomegalovirus(CMV).Methods:Peripheral blood mononuclear cells from four healthy donors were challenged with a 1-week exposure to whole-cell lysate from CMV-infected cells or CMVpp65495-503 peptide(NLVPMVATV).T-cell repertoire perturbations were assessed using the Oncomine TCR Beta-SR Assay and Ion GeneStudio S5 Plus Sequencer.A pp65 tetramer flow cytometry assay was used as an orthogonal method to assess clonal expansion of a subset of CMV-specific T-cells.For evaluation of the assay in peripheral blood lymphocytes from NSCLC donors,five whole blood specimens were evaluated using the same sequencing workflow.Results:The TCR beta assay identified 6,683-61,936 unique clones from 1-2 million reads per sample,and an average of 80%of the total reads were usable for TCR profiling.In the NSCLC donors,TCR convergence and clonality values were consistent with published results and ranged 0.016-0.033 for convergence and 0.09-0.48 for clonality.In the CMV study,TCR sequencing detected the expansion of a common family of clones in all 4 samples in response to antigen stimulation.This expansion corresponded to an increase in pp65 tetramer staining by flow cytometry.Baseline TCR convergence scores ranged 0.009-0.041 and increased 5-fold in one sample as a result of pp65 antigen stimulation.Conclusion:The results of this study demonstrated the utility of profiling of the TCRβrepertoire in a model system and in donors with NSCLC.Additionally,we demonstrated the correlation between RNA-seq methods and protein-tetramer analysis using flow cytometry.These techniques represent an emerging solution that could complement other liquid and tissue diagnostic assays in the clinic and will be of value in predicting host response/resistance and adverse events to immunotherapies.Prospective clinical studies are on-going in which the developed TCR beta assay will undergo further validation。

Circulating tumor DNA in liquid biopsy: Current diagnostic limitation

With the rapid development of science and technology,cell-free DNA(cfDNA)is rapidly becoming an important biomarker for tumor diagnosis,monitoring and prognosis,and this cfDNA-based liquid biopsy technology has great potential to become an important part of precision medicine.cfDNA is the total amount of free DNA in the systemic circulation,including DNA fragments derived from tumor cells and all other somatic cells.Tumor cells release fragments of DNA into the bloodstream,and this source of cfDNA is called circulating tumor DNA(ctDNA).cfDNA detection has become a major focus in the field of tumor research in recent years,which provides a new opportunity for non-invasive diagnosis and prognosis of cancer.In this paper,we discuss the limitations of the study on the origin and dynamics analysis of ctDNA,and how to solve these problems in the future.Although the future faces major challenges,it also con-tains great potential.

Emerging role of liquid biopsy in rat sarcoma virus mutated metastatic colorectal cancer:A case report

BACKGROUND In patients with metastatic colorectal cancer(mCRC),the treatment options are limited and have been proved to be affected by rat sarcoma virus(RAS)mutational status.In RAS wild-type(wt)patients,the combination of antiepidermal growth factor receptor(EGFR)monoclonal antibodies with chemotherapy(CT)is more effective than CT alone.On the other hand,RAS-mutated patients are not eligible for treatment with anti-EGFR antibodies.CASE SUMMARY Eleven patients with initially RAS-mutated mCRC were followed from diagnosis to May 2022.At the time of cell-free DNA determination,five patients had undergone one CT line,five patients had undergone two CT lines,and one patient had undergone three CT lines(all in combination with bevacizumab).At the second and third treatment lines[second line(2L),third line(3L)],patients with neo-RAS wt received a combination of CT and cetuximab.In neo-RAS wt patients treated with anti-EGFR,our findings indicated an increase in progression-free survival for both 2L and 3L(14.5 mo,P=0.119 and 3.9 mo,P=0.882,respectively).Regarding 2L overall survival,we registered a slight increase in neo-RAS wt patients treated with anti-EGFR(33.6 mo vs 32.4 mo,P=0.385).At data cut-off,two patients were still alive:A RAS-mutated patient undergoing 3L treatment and a neo-RAS wt patient who received 2L treatment with anti-EGFR(ongoing).CONCLUSION Our case series demonstrated that monitoring RAS mutations in mCRC by liquid biopsy may provide an additional treatment line for neo-RAS wt patients.

Circulating nucleic acids as liquid biopsies for disease prediction,screening and diagnosis

Liquid biopsy used molecular information in body liquid to perform early diagnosis,screening,monitoring,prognosis,and treatment of various diseases.Circulating free nucleic acids(cfNA)are important diagnostic biomarkers,providing a window to accurately and immediately observe the body's vital activity status.With the development of gene sequencing technology and bioinformatics technology,genetic,epigenetic,and fragtomics alterations that can be detected in cfDNA,as well as the expression level of miRNA and cf-mRNA can be quantified,this can reflect its tissue origin,gene regulation,genome evolution,and disease pathogenesis.This review focuses on the clinical utility of cfNA in different body liquids(blood,urine,bile),and discusses the diagnostic efficacy and accuracy of cfNA as diagnostic biomarkers in a variety of diseases.Blood is widely used to diagnose various tissue lesions for liquid biopsies as a body fluid circulating throughout the body,reflecting the state of the entire body.Bile and urine,as local circulating body fluids,can better reflect the changing state of tissues around the biliary tract and tissues around the bladder,respectively.In addition,normalized sample preservation,cfNA extraction,and detection procedures will help the practical application of cfNA in the clinic.

The combined analysis of solid and liquid biopsies provides additional clinical information to improve patient care

Aim:To investigate if the genetic information provided by sequencing of both solid and liquid biopsies can shed light on tumor heterogeneity,and to understand the clinical usefulness of adding blood profiling to standard tissue analysis in cancer care.Methods:Data from 351 patients with stage IV solid tumors for whom molecular profiling of their solid and liquid biopsies was available were studied,with a focus on the discrepant molecular information found between tissue and blood samples.Results:In 86%of patients,solid and liquid biopsies provided different molecular information.Discrepant gene mutations with a functional impact on the corresponding protein were studied in detail.In 97%of cases,these additional mutations provided clinical value,mainly predicting sensitivity or resistance to targeted therapies.Specifically,42%of the mutations found only in the liquid biopsy were directly predictive of approved therapies(80%targeted therapies),while 54%were inclusion criteria for molecularly-matched trials.Conclusion:This study suggests that the addition of blood profiling should be considered in routine clinical oncology,especially for patients with metastatic disease where integrated analysis of solid and liquid biopsies provides a more complete characterization of tumor heterogeneity and provides valuable clinical information for patient treatment.

Profiling of circulating tumor cells in liquid biopsies from metastatic cancer patients

Aim:Circulating tumor cells(CTCs)are crucial to tumor metastasis and valuable for prediction of clinical outcome in patients with solid tumors.Here,the authors aimed to establish a method for enumeration and characterization of CTCs from liquid biopsies.Methods:Peripheral blood mononuclear cells(PBMCs)were separated from blood samples from patients with metastatic cancer using Ficoll-Hypaque gradients and cultured to isolate and enumerate CTCs.Cultured CTCs were morphologically characterized by light and phase contrast microscopy.The tumorigenicity of Ficoll-Hypaque-separated PBMCs was examined,in addition to their expression of mRNA metastasis markers.Results:CTCs were isolated in culture and enumerated by counting under phase contrast microscopy,demonstrating that 0.01-0.04%of total PBMCs were CTCs.CTCs were dormant,with large,oval-shaped,spiky morphology.PBMCs obtained from liquid biopsies exhibited anchorage-independent growth,forming numerous colonies in soft agar assays.Molecular profiling demonstrated expression of several metastatic genes,but not of cadherin 1(encoding the adhesion protein),in all patients.Conclusion:The authors successfully isolated,enumerated,and characterized CTCs from liquid biopsies of metastatic cancer patients.This study has potential to facilitate the development of new diagnostic and therapeutic methods using liquid biopsies,for application in metastatic cancers.

Liquid biopsies in primary and secondary bone cancers

Liquid biopsies are a powerful tool to non-invasively analyze tumor phenotype and progression as well as drug resistance.In the bone oncology field,liquid biopsies would be particularly important to develop,since standard biopsies can be very painful,dangerous(e.g.,when found in proximity to the spinal cord),and hard to collect.In this review,we explore the recent advances in liquid biopsies in both primary(osteosarcoma and Ewing sarcoma)and secondary bone cancers(breast,prostate,and lung cancer-induced bone metastases),presenting their current role and highlighting their unexpressed potential,as well as the barriers limiting their possible adoption,including costs,scalability,reproducibility,and isolation methods.We discuss the use of circulating tumor cells,cell-free circulating tumor DNA,and extracellular vesicles for the purpose of improving diagnosis,prognosis,evaluation of therapy resistance,and driving therapy decisions in both primary and secondary bone malignancies.

Liquid biopsies to predict CDK4/6 inhibitor efficacy and resistance in breast cancer

Cyclin-dependent kinase 4 and 6(CDK4/6)inhibitors combined with endocrine therapy have transformed the treatment of estrogen receptor-positive(ER+)and human epidermal growth factor receptor 2 negative(HER2-)metastatic breast cancer.However,some patients do not respond to this treatment,and patients inevitably develop resistance,such that novel biomarkers are needed to predict primary resistance,monitor treatment response for acquired resistance,and personalize treatment strategies.Circumventing the spatial and temporal limitations of tissue biopsy,newly developed liquid biopsy approaches have the potential to uncover biomarkers that can predict CDK4/6 inhibitor efficacy and resistance in breast cancer patients through a simple blood test.Studies on circulating tumor DNA(ctDNA)-based liquid biopsy biomarkers of CDK4/6 inhibitor resistance have focused primarily on genomic alterations and have failed thus far to identify clear and clinically validated predictive biomarkers,but emerging epigenetic ctDNA methodologies hold promise for further discovery.The present review outlines recent advances and future directions in ctDNA-based biomarkers of CDK4/6 inhibitor treatment response.

Liquid biopsies in myeloid malignancies

Hematologic malignancies are the most common type of cancer affecting children and young adults,and encompass diseases,such as leukemia,lymphoma,and myeloma,all of which impact blood associated tissues such as the bone marrow,lymphatic system,and blood cells.Clinical diagnostics of these malignancies relies heavily on the use of bone marrow samples,which is painful,debilitating,and not free from risks for leukemia patients.Liquid biopsies are based on minimally invasive assessment of markers in the blood(and other fluids)and have the potential to improve the efficacy of diagnostic/therapeutic strategies in leukemia patients,providing a useful tool for the real time molecular profiling of patients.The most promising noninvasive biomarkers are circulating tumor cells,circulating tumor DNA,microRNAs,and exosomes.Herein,we discuss the role of assessing these circulating biomarkers for the understanding of tumor progression and metastasis,tumor progression dynamics through treatment and for follow-up.

Liquid biopsies in pancreatic cancer:targeting the portal vein

Pancreatic cancer is a highly lethal malignancy with poor overall survival due to silent progression until primary tumor growth or metastatic dissemination develops clinical symptoms.Even in the minority of patients with early diagnosis and candidacy for curative intent surgery,postoperative recurrence after surgical resection is very frequent.Due to these findings,efforts to identify minimally invasive ways to provide earlier diagnosis and enhanced prognostication are increasingly warranted.Liquid biopsies assessing for tumor derived materials shed into the blood are a promising tool to accomplish this goal;however,in pancreatic cancer,peripheral blood analyses remain dependent on the degree of tumor burden with a prohibitively low yield until the cancer is widely metastatic.To overcome this limitation,increasing literature has emerged evaluating the possibility of portal venous blood as a new,potentially higher yield liquid biopsy target in pancreatic cancer.This review will discuss the current literature and clinical application potential of mesenteric vasculature,or portal venous blood,as liquid biopsies in the diagnosis,prognosis and management of patients with pancreatic cancer.

The implication of liquid biopsies to predict chemoresistance in pancreatic cancer

Pancreatic cancer is one of the most aggressive diseases among solid tumors.Most patients are diagnosed with advanced or metastatic disease and are characterized by poor chemosensitivity.Therefore,earlier diagnosis and novel therapeutic possibilities for pancreatic cancer patients are urgently needed.Liquid biopsy is an emerging technology that allows the noninvasive sampling of tumor material.Nowadays,liquid biopsy has shown promising results as diagnostic,prognostic and predictive biomarkers,but it has not yet been universally adopted into regular use by clinicians.In this review,we describe different components of liquid biopsy,especially circulating tumor cells,circulating tumor DNA and exosomes and their potential clinical utility for pancreatic cancer patients.

Liquid biopsy for gastric cancer:Techniques,applications,and future directions

After the study of circulating tumor cells in blood through liquid biopsy(LB),this technique has evolved to encompass the analysis of multiple materials originating from the tumor,such as nucleic acids,extracellular vesicles,tumor-educated platelets,and other metabolites.Additionally,research has extended to include the examination of samples other than blood or plasma,such as saliva,gastric juice,urine,or stool.LB techniques are diverse,intricate,and variable.They must be highly sensitive,and pre-analytical,patient,and tumor-related factors significantly influence the detection threshold,diagnostic method selection,and potential results.Consequently,the implementation of LB in clinical practice still faces several challenges.The potential applications of LB range from early cancer detection to guiding targeted therapy or immunotherapy in both early and advanced cancer cases,monitoring treatment response,early identification of relapses,or assessing patient risk.On the other hand,gastric cancer(GC)is a disease often diagnosed at advanced stages.Despite recent advances in molecular understanding,the currently available treatment options have not substantially improved the prognosis for many of these patients.The application of LB in GC could be highly valuable as a non-invasive method for early diagnosis and for enhancing the management and outcomes of these patients.In this comprehensive review,from a pathologist’s perspective,we provide an overview of the main options available in LB,delve into the fundamental principles of the most studied techniques,explore the potential utility of LB application in the context of GC,and address the obstacles that need to be overcome in the future to make this innovative technique a game-changer in cancer diagnosis and treatment within clinical practice.