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Liquid biopsy in oncology


Solid tumors are made up of multiple subclonal populations of tumor cells, which compete under selective pressures leading to the emergence of dominant subclones that replicate and form the molecular landscape of solid tumors. The genomic landscape of tumors is heterogeneous and longitudinal sampling is essential for precision medicine. This can be achieved with sampling tumor components, such as circulating cell-free DNA (ctDNA), tumor-derived RNA and circulating tumor cells (CTCs), via patients’ blood samples, a process known as ‘liquid biopsy’. The application of liquid biopsies has developed at a high rate over the last 5 years, with over 60 clinical trials and more than 20,000 patients over 11 cancer types evaluated. Exploiting approaches in liquid biopsy can provide a comprehensive overview of tumor characteristics, including aggressiveness and the overall molecular landscape to improve clinical outcomes for patients with cancer. This collection of recent high-quality full-text articles from Springer Nature provides some of the most impactful recent work in this burgeoning area.

Role of liquid biopsy in oncology

Integrating liquid biopsies into the management of cancer

This review evaluates the various types of tumor-derived material which can be sampled using liquid biopsies, which can be integrated into clinical practice to guide patient care. The primary focus is on cell-free tumor DNA (cfDNA), which presently has the greatest clinical application.

  • Due to the heterogeneity of patients with tumors and tumor-specific molecular profile, treatment choices should take these factors into consideration and patient selection is crucial for the success of targeted therapy. Furthermore, the genetic profile of individual tumors is heterogeneous and can change over time due to Darwinian clonal evolution.
  • Presently, tissue biopsies are used to establish the molecular landscape of solid tumors; however, tissue-based profiles are subject to sampling bias and cannot be obtained repeatedly over a long period of time, and are not suitable for longitudinal monitoring of clonal evolution.
  • Tumor-derived materials, such as circulating cfDNA, circulating tumor cells (CTC) and exosomes, are present in the blood of patients with cancer. The sampling and analysis of these components, particularly cfDNA, has enabled a minimally-invasive method of monitoring the dynamic evolution of cancers in the clinic.
  • Liquid biopsy of other body fluids, such as urine, saliva and cerebral spinal fluid, is possible and the success of cfDNA detection is associated with the localisation of the primary tumour and any metastatic lesions.
  • Clinical applications of liquid biopsies include those for diagnostic purposes, identification and tracking of tumour-specific alterations during disease progression and to guide therapeutic decisions. For successful clinical implementation, standardised procedures need to be defined and validated.
  • Currently, regulatory agencies in Europe and the USA have approved two liquid biopsy companion diagnostic tests for EGFR mutation in plasma cfDNA for patients with non-small-cell lung cancer in clinical practice.

Siravegna G et al. Nat Rev Clin Oncol 2017; 14: 531–548. doi: 10.1038/nrclinonc.2017.14

Emerging concepts in liquid biopsies

This article briefly evaluates the current technologies of liquid biopsy in cancer management and the detection rates in the context of mutations in healthy individuals, adjusted for age. The review also discusses emerging new concepts, current challenges in the application of liquid biopsy, as well as, the reliability and clinical applicability of liquid biopsies.

  • Liquid biopsies that analyze circulating tumor DNA (ctDNA) utilize two main approaches, targeted and untargeted. The former is used to analyze single nucleotide mutations or structural chromosomal rearrangements in predetermined genomic regions of plasma DNA to estimate the allelic frequency of a particular mutation. Untargeted approaches rely less on prior knowledge and aim at a comprehensive analysis of the tumor genome.
  • Whole tumor cells in the blood stream, known as circulating tumor cells (CTC), are another approach to liquid biopsy, which utilizes pure tumor DNA or RNA. Single CTC analyses, made possible by improvements of next-generation sequencing technologies and isolations methodologies, can provide evidence of genetic heterogeneity at the individual cellular level.
  • Exosomes are circulating vesicles which contain nucleic acids shed by living cells, including tumors. As they are stable carriers of DNA, RNA and protein from the cell of origin, they are a useful biomarker of cancer. Exosomes also have the potential to detect early stages of cancer if they carry specific mutations.
  • Somatic mutations occur in healthy individuals, often without exhibiting any disease phenotype. Therefore, cancer-associated mutations may be identified in the plasma DNA of healthy individuals. New technologies are required to address this issue.
  • New liquid biopsy technologies include molecular barcodes, which improves low-frequency allele detection, a major challenge with the analysis of ctDNA; plasma RNA analysis, epigenetic signatures and the development of novel plasma DNA and exosome protocols are also concepts that can address the current challenges for liquid biopsy.
  • Before liquid biopsies can be clinically viable diagnostic assays, processes such as pre-analytical and analytical steps require standardization to ensure reproducible processing procedures. Another imperative step is to evaluate the clinical relevance of ctDNA at different time points. Finally, the clinical performance of assays must satisfy the requirements of regulatory agencies.

Perakis S, Speicher M. BMC Med 2017; 15: 75. doi: 10.1186/s12916-017-0840-6

Liquid biopsies come of age: Towards implementation of circulating tumor DNA

In this review, the potential for liquid biopsy is highlighted, with a specific emphasis on approaches in circulating tumor DNA (ctDNA) analysis. Although proof-of-principle studies have demonstrated the clinical potential of ctDNA in determining prognosis, molecularly profiling and monitoring of patients with cancer, there is still much to learn about ctDNA. This review gives a brief overview of ctDNA biology and focuses on the evaluation of potential methods for ctDNA analysis. The application of ctDNA analysis in personalized oncology and cancer research is also evaluated.

  • In clinical oncology, the main areas where liquid biopsies are needed include cancer diagnostics, assessment of prognosis, residual disease and risk of relapse, treatment selection and the monitoring of disease burden.
  • ctDNA is released from cells through apoptosis and necrosis and the genetic and epigenetic modifications of ctDNA molecules reflect the genome of the cell of origin. Sequencing of ctDNA, combined with histological analysis of corresponding tissue samples can provide new insights into the biological determinants of ctDNA fragmentation and its biological origins.
  • The breadth of ctDNA ranges from interrogation of single-locus assays to sequencing the whole genome. Targeted sequencing also allows evaluation of multiple loci with high sensitivity.
  • The concentration of ctDNA in plasma is correlated with tumor size and stage; therefore, it may be useful for determination of cancer stage and prognosis. Of particular importance is the diagnosis of cancer in the early stages of disease, which is associated with improved prognosis and a higher chance of cure. The implementation of ctDNA screening in at-risk populations for earlier diagnosis of disease may be considered, although the risk of over-diagnosis and false positives may occur.
  • Results from liquid biopsies can give information on the tissue of origin and aid cancer localization.
  • Other applications of ctDNA analysis include longitudinal monitoring (for response and recurrence), clonal evolution and resistance.
  • Currently, the proof-of-principle research around ctDNA has demonstrated its usefulness in drug development and for the study of intratumoral heterogeneity and clonal evolution. In future, a better understanding of the origin and biology of ctDNA will aid the implementation of liquid biopsies and randomized trials comparing ctDNA again the usual standard of care will be crucial to inform safety and efficacy in cancer patients.

Wan J et al. Nat Rev Cancer 2017; 17: 223–238. doi: 10.1038/nrc.2017.7

Platelet RNA signatures for the detection of cancer

The role of platelets have evolved from the well-established role in hemostasis and thrombosis, it is now recognized that platelets are implicated in tumor biology and metastasis. Platelets are able to ingest RNA molecules during circulation and possess RNA processing mechanisms. The biological processes in which platelet RNA biomarker signatures can be altered in cancer disease progression is discussed in this review, as well as the role of tumor-educated platelets (TEPs) in liquid biopsies for cancer management.

  • Tumor cells can directly and indirectly affect platelet RNA and protein content, which results in the formation of TEPs. The altered functions of TEPs can promote tumor cell survival and metastasis and can also alter cancer cells to evade immune system detection and physically protect cancer cells from cell mediated immune detection to promote cancer cell survival in the systemic circulation.
  • The potential for utilizing platelets in cancer diagnosis are due to two distinct mechanisms: 1) platelet education arises from the transfer of tumor-associated biomolecules to platelets, and 2) specific splice events in response to external signals and the ability of platelets to ingest circulating mRNA provides TEPs with a dynamic repertoire.
  • TEPs RNA biomarker signatures are altered in the presence of cancer, which can be utilized for liquid biopsy.
  • The future of liquid biopsies relies on an interdisciplinary approach to combine TEPS with other biosources to enable next generation testing for accurate detection and specification of cancer.
  • Several clinical trial protocols have implemented the use of platelet RNA analysis in the early detection of cancer. In particular, the PLATO-VTE study is investigating early detection of cancer in patients with unprovoked symptomatic pulmonary embolism; platelet RNA will be evaluated to determine the cure rates of patients in early detection.
  • Strong quantitative computational analysis is essential to the success of utilizing platelet RNA signatures for cancer detection. The combination of isolation procedure of extracellular vesicles and circulating tumor DNA (ctDNA), TEPs and ctDNA and circulating tumor cells and TEPS should be considered as next-generation biomarker strategies.

Sol N, Wurdinger T. Cancer Metast Rev 2017; 36: 263–272. doi: 10.1007/s10555-017-9674-0

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors

This review evaluates the applicability of comprehensive cell-free DNA (cfDNA) profiling in patients with cancer and demonstrates high concordance of cfDNA; methods for the identification of patients suitable for whole-exome sequencing (WES) is also summarized, with a focus on the feasibility for reproducible and scalable profiling of whole cancer exomes from cfDNA.

  • Comprehensive profiling of tumors from blood may be achieved by WES of cfDNA; however, the genome-wide concordance between cfDNA and tumor biopsies is limited in quantity and comprehensiveness. Furthermore, the ability of cfDNA to capture the genetic diversity of cancer has yet to be systematically explored. Therefore, the application of WES of cfDNA in patients with metastatic cancer is unknown.
  • Previous approaches to screening cancer-associated cfDNA have utilized targeted detection of somatic single nucleotide variants in recurrently mutated cancer genes; however, the majority of metastatic cancers have arm-level somatic copy number alterations, therefore detection of this may be more applicable.
  • Standard depths of WES can be used to detect somatic alterations in tumor samples with 5–10% of tumor content, with reasonable sensitivity. However, due to the variability in cfDNA tumor fractions, advanced screening for tumor content is necessary for the scale-up of WES of cfDNA.
  • ichorCNA is an analytical approach to determine cfDNA samples with sufficient tumor content (>10%) for WES. Analysis of WES of cfDNA and matched tumor biopsy has shown cfDNA to be a suitable proxy for a tumor biopsy. Furthermore, examination of blood samples of patients with metastatic breast or prostate cancer using ichorCNA showed >30% of blood samples, and >40% of patients to have sufficient tumor fraction for standard depths of WES of cfDNA.
  • Major barriers which have been overcome in WES of cfDNA as a method for routine screening include: 1) applicability and efficient screening of tumor content prior to WES for patients with metastatic cancer; 2) comprehensive benchmarking of cfDNA and conventional biopsies.
  • The feasibility of profiling cfDNA for patients with early stage disease requires further investigation; however the ability to detect somatic copy number alterations and estimate tumor content may have broader applicability in routine clinical monitoring of metastatic cancer.

Adalsteinsson V et al. Nat Commun 2017; 8: 1324. doi: 10.1038/s41467-017-00965-y

Role of liquid biopsy in the diagnosis of breast cancer

Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging

Radiology measurements of estrogen receptor positive breast cancer are limited for the assessment of tumor burden, which is essential in the improvement of clinical management of patients. This study evaluates the feasibility of array-based digital polymerase chain reaction (dPCR) to detect and quantify tumor specific mutations of the PIK3CA mutant allele in plasma samples and tumor response.

  • The PIK3CA gene encodes the p110α catalytic subunit of the PI3K protein, and is mutated in more than one-third of breast cancer cases, thus it is an attractive target for tumor monitoring by tumor-specific mutation quantification in plasma samples.
  • There is good agreement between PIK3CA mutation status in patients with advanced cancer and plasma samples; however, in early stage patients the consistency of findings is lower. Indeed, it was reported that PIK3CA mutations were detected in only 22.7% of serum samples from early stage breast cancer patients.
  • This study found correlation between the levels of PIK3CA mutations with treatment responses, according to RECIST criteria, in the majority of patients with breast cancer.
  • In patients where correlation between the levels of PIK3CA mutations with treatment responses was variable, it is likely that tumor heterogeneity in advanced disease was the cause, as evidenced in radiology evaluations.
  • Findings from this study suggest that information provided by oncogenic mutations in plasma samples is limited by tumor heterogeneity in advanced disease. Results should be evaluated in conjunction with radiographic imaging.

García-Saenz J et al. BMC Cancer 2017; 17: 210. doi: 10.1186/s12885-017-3185-9

Liquid biopsy in breast cancer

In this chapter, genomic profiling in breast cancer is introduced, as well as the combined use of clinicopathological information and genomic profiling to estimate the risk of recurrence and identify high-risk patients with breast cancer to inform treatment strategies for disease subgroups.

  • Tumor heterogeneity in patients with breast cancer arises from spatial and temporal intra-tumoral heterogeneity as well as inter-tumoral heterogeneity. This phenomenon is one of the main barriers to precision medicine in breast cancer. In order to obtain real-time molecular information of tumors, the non-invasive technique of liquid biopsy can be utilized to follow the evolving genomic landscape.
  • Levels of circulating tumor DNA (ctDNA) is correlated to tumor size, lymph node involvement, histopathological grade and clinical staging. ctDNA can be used in clinical practice for the evaluation of diagnosis, treatment and management of patients with breast cancer.
  • PIK3CA is one of the main genes involved in tumorigenesis of breast cancer. PIK3CA mutation was detectable in ctDNA in women with stage 1–3 breast cancer with a frequency of 23% overall, and 80% in patients with metastatic cancer.
  • Treatment efficacy can be monitored using ctDNA. A decrease in ctDNA concentrations was found after surgery and chemotherapy, suggesting that ctDNA can act as a marker of treatment response.
  • In breast cancer management, one of the main issues is testing the feasibility of liquid biopsies in the evaluation of minimal residual disease (MRD), which is defined as the lowest level of residual disease after curative therapy. The ability to detect MRD after first-line treatment can inform adjuvant treatment to avoid disease recurrence.
  • Circulating tumor cells (CTCs) may be useful to improve clinical outcomes in triple-negative breast cancer. Increased CTC number after new treatment is correlated with shortened progression-free survival/overall survival and overall to poorer prognosis.

Incorvaia L et al. In: Liquid Biopsy in Cancer Patients. Edited by Russo A, Giordano A, Rolfo C. Humana Press, Cham, 2017. doi: 10.1007/978-3-319-55661-1_9

Role of liquid biopsy in the diagnosis of head and neck cancer

Tumor DNA: An emerging biomarker in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the most common form of head and neck cancer. Patients often present with advanced disease and HNSCC has a 5-year survival rate of ~50%. Tissue biopsy with cytopathology or histopathology is the mainstay for diagnosis of HNSCC; however, these conventional methods are temporally and spatially limited and only provide a snapshot of a single region of a heterogeneous tumor. The use of liquid biopsy to analyze tumor DNA, an emerging biomarker of HNSCC, is minimally invasive and can be used for tumor profiling, and in determining prognosis and guide management decisions.

  • Currently, due to the paucity of disease-specific biomarkers, treatment decisions for HNSCC are made based on conventional imaging, histological evaluations and clinical examination to evaluate disease burden, response to therapy and likelihood of recurrence. Due to the location of HNSCC, diagnostic procedures present anatomic challenges, which may lead to delays in treatment and negative effects in prognosis and survival.
  • The changing paradigm in HNSCC management is focusing on early and accurate diagnosis and molecular characterization. Advances in gene sequencing have facilitated the detection of mutated DNA from bodily fluids.
  • HNSCC is by definition a mucosal disease in which all tumors are anatomically related to mucosal surfaces. Therefore, tumor DNA can be collected from the basal surface of a tumor via the blood and from the apex via saliva.
  • Tumor DNA of HNSCC is determined by the presence of somatic mutations (TP53, PIK3CA, CDKN2A, NOTCH1) and HPV viral genes (E6, E7), which are highly specific when isolated from saliva and plasma samples.
  • Tumor DNA appears to be more detectable in early HNSCC, compared with other tumor types.
  • The future utility of tumor DNA allows accurate characterization of individual tumors, which can open the door to a new generation of targeted and personalized therapeutic management. Better disease monitoring and surveillance can be achieved with improving techniques to quantify tumor DNA.

Bellairs J, Hasina R, Agrawal N. Cancer Metastasis Rev 2017; 36: 515–523. doi: 10.1007/s10555-017-9685-x

Role of liquid biopsy in the diagnosis of melanoma

Liquid biopsies in malignant melanoma: From bench to bedside

Melanoma originates from melanocytes and is a neoplastic disorder with an increasing incidence in the last decade. Different pathways have an active role in distinct subtypes of melanoma. The fundamental pathways of tumorigenesis for cutaneous melanoma are reviewed in this chapter.

  • The mitogen-activated protein kinases (MAPK), PI3K/AKT, p16 (INK4a)-Rb pathways have been investigated in the tumorigenesis of melanoma. c-KIT receptor mutations are also associated with melanoma, although rarely (10%); they are associated with activation of MAPK pathway.
  • Immunotherapy is the best treatment option for metastatic melanoma and depends on factors such as the history of disease, symptoms and BRAF mutation status. Interferon (IFN)-α 2b and pegylated IFN-α 2b have shown clinical benefit as an adjunctive treatment to surgery. Other monoclonal antibodies, such as ipilimumab and pembrolizumab, have also shown clinical benefit in the treatment of melanoma.
  • In the clinical management of melanoma, dacarbazine is considered standard chemotherapy for advanced disease and is associated with a median overall survival of 6–7 months.
  • Targeted therapies utilizing the MAPK or RAS/RAF/MEK/ERK signaling pathway, which regulates cell growth, proliferation and differentiation, are also used to treat melanoma.
  • BRAF mutations activate the downstream signal transduction of the MAPK pathway. BRAF inhibitors, such as vemurafenib and dabrafenib, are able to target BRAF-mutant melanoma cells and inhibit cell growth and proliferation.
  • BRAF inhibitors can be used in conjunction with MEK inhibitors, which blocks reactivation of the MAPK pathway downstream from BRAF, and is able to overcome issues of resistance to BRAF inhibitors.
  • Melanoma cells are able to release a number of substances into circulation by active secretion or cell death. These biomarkers in melanoma patients may offer a complete profile of information on diagnosis, staging and prognosis and may be suitable for liquid biopsy.

Alegre E et al. In: Liquid Biopsy in Cancer Patients. Edited by Russo A, Giordano A, Rolfo C. Humana Press, Cham, 2017. doi: 10.1007/978-3-319-55661-1_17

Role of liquid biopsy in the diagnosis of lung cancer

Liquid biopsy in non-small cell lung cancer (NSCLC)

Lung cancer is characterized by a high degree of heterogeneity in tumors with different molecular alterations, such as EGFR mutations, ALK-EML4 translocations, and RAS mutation being the most common. Due to genomic instability of tumors, resistance mechanisms will develop and the molecular alterations of tumors will not be homogenously distributed within the same lesion. Furthermore, the molecular profile of metastasis can be completely different to the primary tumor. Therefore, it is particularly important to complete molecular analysis of the tumor and any metastatic lesions. Liquid biopsy analysis can improve the management in patients with NSCLC at different stages in disease progression, from diagnosis to relapse.

  • Tissue biopsy is traditionally used to evaluate the molecular composition of tumors; however, it is limited by its invasiveness, difficulty in accessing tumor sites and detecting heterogeneity. Liquid biopsy, in particular circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes, may be an alternative as it can be easily repeated at different times, allowing continual monitoring of the molecular status of tumors over the treatment course.
  • CTC are shed from both the primary and metastatic tumors and can be used to evaluate the risk of metastasis and acts as a source of nucleic acid for molecular characterization. The technical challenge with CTC is mainly due to the limited performance of isolation and detection methods; however, with novel highly sensitive techniques now available, there is potential for analysis of molecular alterations of CTC using relatively simple methods.
  • To analyze the genomic landscape, new sensitive techniques are required to analyze EGFR mutation status from plasma-derived ctDNA. In particular, digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) platforms are the most studied approaches.
  • The analysis of ctDNA also has diagnostic and prognostic value, where plasma-based EGFT testing in patients with NSCLC showed the same degree of sensitivity and specificity, compared with the gold standard tissue genotyping.
  • Exosomes differ from other components of the liquid biopsy as they are actively released by tumor cells; therefore, they have a potential role in tumor progression. The role of exosomes in NSCLC is promising; however, the lack of consensus in the optimal way to isolate exosomes from body fluids needs to be addressed before implementation into clinical practice.
  • Platelets, with their ability to engulf tumor-related RNA, are currently being investigated for liquid biopsy in patients with NSCLC.

Rolfo C et al. In: Liquid Biopsy in Cancer Patients. Edited by Russo A, Giordano A, Rolfo C. Humana Press, Cham, 2017. doi: 10.1007/978-3-319-55661-1_12

Role of liquid biopsy in the diagnosis of gastrointestinal cancer

Diagnostic and prognostic performance of liquid biopsy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and arises from chronically damaged organ. Although molecular based prognostic predictors have been identified, none have reached clinical practice. Diagnostic biopsies are obtained in less than 20% of patients with HCC, thus reducing the access to tissue for molecular analysis. Studies on liquid biopsy in HCC are limited and the identification of genomic alterations via liquid biopsy is lagging behind molecular analysis from tissue samples. In this chapter, the role of liquid biopsy as a diagnostic and prognostic tool is summarized.

  • Currently, patients at high risk are monitored every 6 months with abdominal ultrasound; if a liver nodule is detected, computed tomography and magnetic resonance imaging are gold standard for HCC diagnosis. Early detection is not always possible as the sensitivity of imaging is limited for small lesions. Liquid biopsy can fulfill this gap and may be utilized as an early detection tool for HCC development.
  • CTC detection is more frequent in patients at advanced stages of HCC and isolation techniques are being researched and validated. Although data on CTCs are promising, further investigation is required to clarify the role in early detection.
  • Methylation changes have been investigated as a potential surrogate marker of HCC development. Genes of interest include RASSF1A, P16 and P15; combining hypermethylation of these genes with other clinical factors is associated with high HCC detection.
  • The worst prognostic factor in HCC is metastatic dissemination to distant organs and results from the spread of malignant cells through the systemic and lymphatic circulations. Therefore, identification of CTC is a potential marker for disease spread.
  • The prognostic value of circulating DNA (ctDNA) is still under investigation; however, a recent study explored the potential diagnostic role and found that aberrant methylation of RASF1A was associated with patient survival rates.
  • The molecular subclasses of liver cancer are currently under investigation to find oncogene addiction loops, which may be used for early diagnosis. Micro-RNA (miRNA) has appealing diagnostic potential. Tissue biopsy is not currently mandatory for diagnosis of liver cancer; however, in the future, the diagnostic and prognostic role of liquid biopsy may heavily influence the management strategies in HCC.

Labgaa I, Craig AJ, Villanueva A. In: Liquid Biopsy in Cancer Patients. Humana Press, Cham, 2017. Edited by Russo A, Giordano A, Rolfo C. doi: 10.1007/978-3-319-55661-1_14

Liquid biopsy in colorectal cancer

There are four molecular subtypes hypothesized for colorectal cancer (CRC) with different characteristics and biological behavior, CMS1 (microsatellite instability), CMS2 (overexpression of WNT and MYC signal-dependent pathways), CMS3 (metabolic dysregulation) and CMS4 (mesenchymal type). Liquid biopsy is able to study features of circulating tumor cells (CTCs), cell-free DNA (cfDNA), exosomes and microRNA (miRNA) to guide management decisions in patients with CRC.

  • CTCs may be useful in selection therapeutic options in adjuvant and metastatic settings through the identification of specific molecular targets, such as cytokeratin-19, cytokeratin-20, carcinoembryonic antigen and human telomerase reverse transcription mRNA. CTC can also help estimate the risk of recurrence in patients who underwent curative surgery.
  • The concentration of circulating cell-free DNA (cfDNA) related to biological characteristics of tumors, such as size and growth rate. Quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) techniques have high sensitivity. Monitoring of minimal residual disease is important in CRC as recurrence is around 30% and 50% in patients with Dukes’ stages 2 and 3 disease; this is likely caused by the presence of micrometastasis.
  • A possible role of cfDNA in CRC is the identification of wild-type RAS mutations, which are effectively treated with panitumumab and cetuximab. The mutation status of KRAS and other genes from the same family are evaluated in patients with CRC as resistance is conferred in patients with positive mutations. This evaluation is routinely performed on tissue samples; however, cfDNA may be a less invasive method to predict in response.
  • Exosomes are another potential neoplastic marker in CRC. At room temperature, exosomes remain stable but their number increases in low-pH environments, a characteristic typical to the cancer microenvironment. At a molecular level, exosomes contain information to modulate cancer-mediated growth pathways by promoting cell-cell communication.
  • Exosomes are divided in a subgroup carrying protein typical of basolateral colonic epithelium region (A33 +) and a subgroup carrying EpCAM +, a protein of the apical epithelium. Exosomes also have the ability to interact with the MHC II complex to exert a role in immune surveillance of the intestinal mucosa.
  • Micro-RNA (miRNA) has been found in greater numbers in exosomes isolated from CRC cell lines, compared with healthy cells. miRNA can serve as a potential biomarker which modulates oncogenic properties of cells, for example, miR-21 is found to be upregulated in CRC and other malignancies.

Galvano A et al. In: Liquid Biopsy in Cancer Patients. Edited by Russo A, Giordano A, Rolfo C. Humana Press, Cham, 2017. doi: 10.1007/978-3-319-55661-1_13

Role of liquid biopsy in the diagnosis of urologic cancer

Liquid biopsy: A step forward towards precision medicine in urologic malignancies

The exploration of minimally invasive tests to identify biomarkers in cancer has extended to urologic malignancies. The detection of circulating cell-free DNA (cfDNA), circulating tumor cells (CTCs), circulating RNAs, cell-free proteins, peptides and exosomes in liquid biopsies hold promise for personalized medicine due to ability to provide a global snapshot of primary and metastatic tumors. This review provides an overview of the current research in cfDNA based biomarkers in kidney, prostate and bladder cancers and discuss the potential utility of other circulating molecules.

  • In patients with renal cell carcinoma (RCC), bladder and prostate cancers, cfDNA is detectable in at least 50% of serum samples and over 70% of urine samples. However, tumor-specific mutations can be as low as 0.01% of total cfDNA and sensitive methods of detection and quantification are required.
  • Results from the evaluation of cfDNA characteristics, such as circulating levels, integrity, methylation and mutation status, can give rise to clinical utility in the management of kidney, bladder and prostate cancers.
  • Multiples studies have shown that plasma cfDNA levels are higher in patients with cancer, compared with control subjects, with high sensitivity and specificity. For example, it was found that cfDNA levels were lower in patients with RCC in remission, compared with those with progressive disease. Higher cfDNA levels during the treatment also indicated poor prognosis.
  • The integrity of cfDNA is a measure of the extent of cfDNA fragmentation, calculated by the ratio of long-to-short cfDNA fragments derived from necrotic and apoptotic cells. Highly fragmented DNA is usually derived from apoptotic cells, whereas longer DNA fragments result from necrotic cancer cells. Patients with RCC had elevated cfDNA integrity, compared with healthy individuals, suggesting the presence of DNA from necrotic cancer cells.
  • In another study, it was found that CpG island hypermethylation of serum cfDNA occurred more frequently in patients with RCC relative to healthy subjects and was able to diagnose RCC with high 63% sensitivity and 87% specificity.
  • Serum CTC levels were raised in patients with advanced stage RCC and were associated with a more aggressive phenotype. Peripheral CTC levels were also associated with lymph node status and the presence of metastasis.
  • Circulating RNAs, including messenger RNA, microRNA and long non-coding RNA have recently been recognized as cancer biomarkers. Despite its potential, the translation into clinical practice is limited due to the lack of stability and inter-individual variability of RNA in circulation.
  • Circulating proteins and peptides are a relatively new cancer biomarker and several non-invasive multi-marker tests are commercially available. Exosomes are also a potential source of cancer biomarkers; however the lack of accurate isolation and detection methods has hindered their use in clinical practice.

Di Meo A et al. Mol Cancer 2017; 16: 80. doi: 10.1186/s12943-017-0644-5

Exosomal microRNAs in liquid biopsies: Future biomarkers for prostate cancer

Prostate cancer is diagnosed based on plasma quantification of prostate-specific antigen, but lacks specificity. Micro-RNAs (miRNAs) are non-coding RNA that regulate gene expression and are present in cell-derived extracellular vesicles, such as exosomes. This review summarizes the potential utility of exosomal miRNA in prostate cancer.

  • A series of complex genetic and environmental influences contribute to the malignant progression of prostate epithelial cells and progression to carcinoma. Prognostic markers currently used are serum prostate specific antigen (PSA) levels, Gleason score and pathological stage. Other than the invasive produce of a prostate biopsy, no tests are able to determine the aggressiveness of the tumor or its metastatic potential.
  • miRNA expression in tumor has been found to have different regulation behavior, compared with normal tissue, supporting their complex dual role either as oncogene or tumor suppressor gene. For example, miRNA-125b is an oncomir in prostate cancer but acts as a tumor suppressor in human ovarian and breast cancer.
  • Exosomes are made up of messenger RNA, miRNA and DNA so the transfer of information and oncogenic signaling to the tumor microenvironment allows for modulation of tumor progression, proliferation, angiogenesis and the suppression of immune responses.
  • Prostasomes are microvesicles found inside vesicles of prostate epithelial cells and are present in prostate secretions. The levels of prostasomes are reportedly increased in patients with prostate cancer and are associated with disease aggressiveness.
  • A proteome study found that urinary exosomes in patients with prostate cancer had 246 proteins which were differentially expressed, compared with healthy male controls.
  • A urinary 3-gene expression assay for exosomes had improved identification of patients with high grade prostate cancer with increased PSA and reduced the number of unnecessary biopsies.
  • An investigation of specific miRNAs found that higher levels of mir-107, mir-574-3p and mir-483-5p were found in the urine of men with prostate cancer, compared with healthy men.
  • Personalized medicine is achievable with the advent of liquid biopsies in prostate cancer; however, the development of new exosome isolation methods and high-throughput technologies are needed to substitute the use of exosomal miRNAs, a source of new prostate cancer biomarkers, for invasive procedures of biopsy or serum PSA.

Valentino A et al. Clin Transl Oncol 2017; 19: 651–657. doi: 10.1007/s12094-016-1599-5