A new type of blood test for breast cancer could help avoid thousands of unnecessary surgeries and otherwise precisely monitor disease progression, according to a study led by the Translational Genomics Research Institute (TGen) and Mayo Clinic in Arizona.
TGen is an affiliate of City of Hope, which along with The Cancer Research UK Cambridge Institute at Cambridge University and the Biodesign Institute at Arizona State University (ASU) also contributed to this study.
Published today in the premier journal Science Translational Medicine, the study suggests that the test called TARDIS – TARgeted DIgital Sequencing – is as much as 100 times more sensitive than other blood-based cancer monitoring tests.
TARDIS is a “liquid biopsy” that specifically identifies and quantifies small fragments of cancer DNA circulating in the patient’s bloodstream, known as circulating tumor DNA (ctDNA).
According to the study, TARDIS detected ctDNA in as low as 2 parts per 100,000 in patient blood.
“By precisely measuring ctDNA, this test can detect the presence of residual cancer, and inform physicians if cancer has been successfully eradicated by treatment,” said Muhammed Murtaza, M.B.B.S., Ph.D., Assistant Professor and Co-Director of TGen’s Center for Noninvasive Diagnostics. He also holds a joint appointment on the Research Faculty at Mayo Clinic in Arizona, and is one of the study’s senior authors.
For example, Dr. Murtaza explained, TARDIS is precise enough to tell if early stage breast cancer patients have responded well to pre-operative drug therapy.
It is more sensitive than the current method of determining response to drug therapy using imaging.
“This has enormous implications for women with breast cancer.
This test could help plan the timing and extent of surgical resection and radiation therapy after patients have received pre-operative therapy,” said Dr. Barbara A. Pockaj, M.D., a surgical oncologist who specializes in breast and melanoma cancer patients at Mayo Clinic in Arizona, and is the study’s other senior author. Dr. Pockaj is the Michael M. Eisenberg professor of surgery and the chair of the Breast Cancer Interest Group (BIG), a collaboration between researchers at Mayo, TGen and ASU.
Unlike traditional biopsies, which only produce results from one place at one time, liquid biopsies use a simple blood draw, and so could safely be performed repeatedly, as often as needed, to detect a patient’s disease status.
This study was performed in collaboration with Carlos Caldas, M.D., Professor of Cancer Medicine at the University of Cambridge and Director of the Breast Cancer Programme at the Cancer Research UK Cambridge Cancer Centre.
“TARDIS is a game changer for response monitoring and residual disease detection in early breast cancer treated with curative intent.
The sensitivity and specificity of patient-specific TARDIS panels will allow us to tell very early, probably after one cycle, whether neo-adjuvant (before surgery) therapy is working and will also enable detecting micro-metastatic disease and risk-adapted treatment after completing neo-adjuvant therapy,” said Dr. Caldas, who also is Senior Group Leader at the Cancer Research UK Cambridge Institute, and one of the study’s contributing authors.
Following further clinical testing and trials, TARDIS could someday be routinely used for monitoring patients during cancer treatment, and discovering when patients are essentially cured and cancer free.
“The results of these tests could be used to individualize cancer therapy avoiding overtreatment in some cases and under treatment in others,” Dr. Murtaza said.
“The central premise of our research is whether we can develop a blood test that can tell patients who have been completely cured apart from patients who have residual disease.
We wondered whether we can see clearance of ctDNA from blood in patients who respond well to pre-surgical treatment.”
Current tests and imaging lack the sensitivity needed to make this determination.
“Fragments of ctDNA shed into blood by tumors carry the same cancer-specific mutations as the tumor cells, giving us a way to measure the tumor,” said Bradon McDonald, a computational scientist in Dr. Murtaza’s lab, and the study’s first author.
“The problem is that ctDNA levels can be so low in non-metastatic cancer patients, there are often just not enough fragments of ctDNA in a single blood sample to reliably detect any one mutation.
This is especially true in the residual disease setting, when there is no obvious tumor left during or after treatment,” McDonald said.
“So, instead of focusing on a single mutation from every patient, we decided to integrate the results of dozens of mutations from each patient.”
The study results suggest that personalized ctDNA analysis, using TARDIS, is a promising approach to identifying patients with a curative response following pre-surgical drug therapy.
“Together with imaging and tissue-based predictive biomarkers, ctDNA is rapidly becoming a useful diagnostic test to determine individualized decisions about additional treatment,” Dr. Murtaza said.
Dr. Pockaj affirmed: “We are excited that TARDIS has the potential to really individualize clinical management of patients with non-metastatic cancer.”
Thomas Slavin, M.D., Assistant Clinical Professor at City of Hope National Medical Center, and a contributing author of the study, noted that “reliably identifying, often multiple, circulating tumor mutations in the plasma of patients with non-metastatic breast cancer also holds promise that ctDNA may one day be a great tool for early breast cancer detection.”
TGen is now focused on evaluating the best partners to work with to automate and scale TARDIS, so it can be made available broadly to benefit patients in need.
“This data represents an exciting strategy to improve the sensitivities of liquid biopsies, which have been challenging for breast cancer,” said Karen Anderson, M.D., Ph.D., a researcher at the Biodesign Institute, a medical oncologist at Mayo Clinic in Arizona, and one of the study’s contributing authors.
“This work represents highly collaborative efforts across multiple institutions, and with the generosity and foresight of our patients who have contributed to this study.”
Cancer is one of the greatest threats facing our society, being the second leading cause of death globally.
Currents strategies for cancer diagnosis consist of the extraction of a solid tissue from the affected area.
This sample enables the study of specific biomarkers and the genetic nature of the tumor.
However, the tissue extraction is risky and painful for the patient and in some cases is unavailable in inaccessible tumors.
Moreover, a solid biopsy is expensive and time consuming and cannot be applied repeatedly.
New alternatives that overcome these drawbacks are rising up nowadays, such as liquid biopsy.
A liquid biopsy is the analysis of biomarkers in a non-solid biological tissue, mainly blood, which has remarkable advantages over the traditional method; it has no risk, it is non-invasive and painless, it does not require surgery and reduces cost and diagnosis time.
The most studied cancer non-invasive biomarkers are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes.
These circulating biomarkers play a key role in the understanding of metastasis and tumorigenesis, which could provide a better insight into the evolution of the tumor dynamics during treatment and disease progression.
Improvements in isolation technologies, based on a higher grade of purification of CTCs, exosomes, and ctDNA, will provide a better characterization of biomarkers and give rise to a wide range of clinical applications, such as early detection of diseases, and the prediction of treatment responses due to the discovery of personalized tumor-related biomarkers.
The abnormal and uncontrolled cell growth, known as cancer, is considered the second leading cause of death globally. Lately, personalized medicine has been gaining a lot of attention for being one of the most promising areas for cancer therapy.
According to the World Health Organization (WHO), the number of deaths owed to cancer in 2015 was 8.8 million globally and in 2014, almost 1.3 million people died from this disease in the EU (World Health Organization, 2018).
For the EU, the standardized death rate in 2014 was 261.5 per 100,000 inhabitants, which was lower than the rate for circulatory diseases, but higher than the rate for most other causes of death [1].
The effect of cancer on society is massive, and an early, sensitive, and accurate diagnosis can be considered sine qua non in cancer management, as it can lead to effective therapeutic interventions, reducing the treatment cost and substantially improving patient outcome and overall survival (OS) [2].
Currently, clarification of the molecular landscapes of the tumor is crucial to guide and provide better treatment choices in clinical practice.
Tissue biopsies are the current method to access the molecular information of the tumor and are required for the identification of its nature, such as type of cancer, gene and mutation expression, and screening [3].
However, it is fraught with issues, such as a required invasive surgical extraction, which could cause discomfort, pain, and risk for the patient.
There are several clinical risks that are inherent in procedures and the possibility of surgical complications.
Moreover, some tumors are difficult to access in some anatomical locations, which are not accessible for a biopsy; and, in some cases, the extraction of it may augment the risk of metastatic lesions [4].
Sometimes the amount of tissue extracted in not enough for all the required tests and it needs to be repeated, which is also needed if the tumor is not homogeneous and/or is evolving along with the disease.
Moreover, the solid biopsy methodology involves a high financial cost, it is time consuming, and requires an operating theater.
So, even if different metastatic locations could be biopsied simultaneously, there could be a delay in the start of the treatment due to the analysis of the samples and it might compromise the prognosis [5].
Moreover, the evolution of the tumor needs to be monitored at separate times of the disease for an efficient treatment of the illness, so solid biopsies cannot be considered again for being highly invasive, and, normally, optical methods are used; however, they do not provide complete information about the tumor.
Radiology is also widely used; however, excessive levels of radiation could generate a health risk for the patient.
Non-radiation approaches, such as magnetic resonance imaging (MRI) scans, are considered inconclusive and inefficient for the minimal residual disease (MRD) detection (reviewed in detail by [6,7]), and, also, due to the limited information provided.
Moreover, safety can be questionable, for instance, regarding sampling of tumors surrounding major vessels or in eloquent regions of the brain, or in patients with major comorbidities [8].
Therefore, these types of techniques are clinically unfeasible and unable to encompass the temporal and spatial heterogeneity of the tumor, i.e., several populations of cancerous cells (with different genetic variations) might exist in different regions among the tumor spatial heterogeneity or have considerable differences between the original lesion and a recurrence, that could be either a local or distant temporal heterogeneity [2,9,10].
Considering the temporal and spatial heterogeneity of the tumor, the most logical step to follow is the extraction of several biopsies from the patient’s initial lesion and their respective metastasis; however, all the drawbacks discussed previously make this possibility unfeasible.
Consequently, there is an urgent need to search for minimally invasive biomarkers in order to detect and monitor the disease progression at several time-intervals throughout the treatment.
One promising alternative to tissue biopsy is the study of the communication between cancer cells, or any other cancer-related biomolecule (cells, nucleic acids, proteins, microvesicles, etc.) and their microenvironment.
By understanding this approach, researchers and clinicians could be able to foresee the behavior and response of tumor cells to patient-specific therapies, allowing an improvement of outcomes and OS rate.
Liquid biopsy, defined as the capture of tumor-related biomarkers in a fluid sample, has been extensively studied and is growing in popularity due to its minimal invasiveness, low consumption of reagent, and ease-of-use.
During the apoptosis and necrosis of tumor cells, these biomarkers are released into the bloodstream, facilitating and promoting metastatic activity in nearby and/or distant organs. Lately, this approach has been considered for applications in the early diagnosis of tumors, therapeutic guidance, and recurrence monitoring. These advantages are possible due to the abundant information that it can provide [11].
According to several research studies, the liquid biopsy approach has been focused on the analysis of circulating tumor cells (CTCs), circulating tumor nucleic acids (ctNAs) and/or tumor-derived extracellular vesicles (exosomes), which have been shed from tumors, and their metastatic sites, into the bloodstream, saliva, urine, cerebrospinal fluid (CSF), among other peripheral fluids of cancer patients. Figure 1 shows the scheme of the different tumor-related biomarkers aforementioned and their presence in blood—further reviewed in ref. [12].
The concentration of these biomarkers in the bloodstream might contribute to an earlier detection of the stage of cancer and a more favorable prediction of the prognosis in patients.
Schematic of the origin of cell-free DNA (cfDNA), circulating tumor cells (CTCs), and exosomes in the blood by [12], licensed under CC BY-NC-ND. The final, published version of this article is available at http://www.karger.com/?doi:10.1159/000458736.
It has been found that levels of these biomarkers increase in patients with several malignant types of tumors, such as prostate, colorectal, stomach, lung, breast, among others. Most studies have been done in patients with late-stage cancer, usually stage III and IV, mainly due to the considerably higher volumes of the aforementioned biomarkers in their blood.
In patients with prostatic cancer, a study showed that more than 5 CTCs per 7.5 mL of blood [13], and an approximate circulating tumor DNA (ctDNA) concentration of 437 ng/mL of blood vs. healthy patients (99 ng/mL of blood) [14] could be considered unfavorable, so it could reduce considerably their OS. Regarding colorectal cancer, a study revealed a significantly broad range for ctDNA in patients suffering from it (22–3922 ng/mL of blood), in comparison with healthy ones (5–16 ng/mL of blood) [15].
Several gastrointestinal diseases could also lead to an increase in ctDNA, even if considered benign or malignant. Normal controls presented ctDNA levels of 14 ± 3 ng/mL, which are remarkably lower than those obtained for malignant and benign diseases, 412 ± 63 ng/mL and 118 ± 14 ng/mL, respectively (Shapiro et al., 1983). Lung cancer patients and control patients, unlike other types, demonstrate significant differences in ctDNA levels since stage I, i.e., 318 ng/mL and 18 ng/mL respectively [16].
Among other studied types of cancer, women suffering from metastatic breast cancer presented two or more CTCs per 7.5 mL of blood, in comparison to healthy women (0.1 per 7.5 mL of blood) [17].
Even though CTCs and ctDNA can be considered as an attractive tool for early detection and diagnosis of cancer, recent studies have shown that the sensitivity and specificity of tests could be considerably improved if coupled with conventional biomarkers for several types of tumors.
For instance, a test combining ctDNA with carcinoembryonic antigen (CEA) for colorectal cancer, and pancreatic carcinoma, ctDNA with prostate-specific antigen (PSA) for prostate cancer [14], and ctDNA with microsatellite alterations for lung cancer [16], could be a potentially useful tool for a better understanding of the disease progression and early-stage diagnosis.
In this comprehensive review, the source, characteristics, and technology of detection of CTCs, ctNAs, and exosomes, as well as their use in the diagnosis, recurrence monitoring, and prognostic assessment for tumors is described.Go to:
Concept of Liquid Biopsy
In order to properly discuss the liquid biopsy approach in detail, it is pivotal to understand the different types of cancer-related biomarkers and their respective biogenesis. For several years, CTCs, extracellular vesicles called exosomes, and ctNAs, such as ctDNA and microRNAs (miRNAs), have been considered the main biomarkers in the liquid biopsy approach for cancer [18].
As mentioned before, these biomarkers are shed off into peripheral fluids from the tumor site, so they can be detected and analyzed in order to improve the clinical settings of a patient tumor.
Indeed, the rapid turnover of cancer cells is assumed to be the cause of the constant release of tumor-derived nucleic acids, vesicles, and viable CTCs into the circulation. Thus, the ability to detect and characterize circulating cell-free tumor DNA (ccftDNA), tumor-derived RNA (predominantly microRNAs (miRNAs)), and CTCs, has enabled doctors and surgeons to analyze the evolution of the tumor several times and, most importantly, in a non-invasive approach.
Circulant Tumor Cells (CTCs)
Biogenesis
While examining peripheral blood under a microscope, Thomas Ashworth discovered CTCs in 1860. Therefore, a theory based on the penetration of tumor cells into the vessel wall and their respective entry in the bloodstream was then proposed [19].
Particularly, most CTCs were found to be accidental circulant cells, i.e., being passively or actively pushed by external forces, including tumor growth, and mechanical stress during surgical operation [20].
CTCs are, indeed, tumor cells that are mostly shed from primary lesions during their formation and early growth.
They circulate through the bloodstream to potential metastatic sites, either as a single cell or in clusters, becoming the main mechanism for metastasis [21,22].
In order to capture and analyze CTCs, it is important to understand their major obstacles: Their complex surface and overall heterogeneity make them extremely rare. One of them is that they are, quite literally, one in a million or billion (approx. 1 cell per 109 blood cells in patients with metastatic cancer), among other cells in the blood [8].
Another challenge has to do with the variety of their surface protein expressions, sizes, and physical characteristics, depending on the type and stage of cancer. Additionally, the number of captured and detected cells in the blood of patients with any type of cancer has been correlated with treatment outcomes and OS.
The capture of these biomarkers could be done in several ways, including immuno-recognition, separation based on size or stiffness, among other chemical or physical recognition methods.
Technologies and Strategies for Detection
The main challenge in the detection of CTCs is the identification and characterization of single tumor cells, which are remarkably hard to locate, compared to the millions of other hematopoietic cells.
As mentioned before, its rarity makes it extremely difficult, along with its low abundance and concentration, and even more in early-stage cancers, as it is at even lower concentrations.
Therefore, it requires extremely sensitive and specific analysis methods, which have been developed in order to achieve maximum CTC collection. The enrichment and detection methods of CTCs can be classified based on their biological and physical properties, as Figure 2 describes below.
Biological and physical approaches of enrichment. Retrieved with permission from [23]. Copyright 2016, Elsevier.
Biological Properties
Detecting CTCs based on their biological properties is mostly related with proteins present in the cell membrane.
To target these molecules immunoselection-based procedures are used, with two different approaches: Antibodies against tumor-associated antigens, which is called positive selection, or against the common leukocyte antigen, which is called negative selection.
A very recognized technique is the immunomagnetic isolation, which consists of the attachment of an antibody to a magnetic bead in order to target an antigen.
The formed antigen–antibody complex can then be isolated by exposing the sample to a magnetic field. Based on several studies, antibodies against the epithelial cell adhesion molecule (EpCAM) are commonly used for positive selection.
CellSearch is the only EpCAM-based assay that is FDA-approved, and it is also considered the “gold standard” for CTC detection.
As mentioned, the main goal of this technique is to enumerate epithelial CTCs biomarkers, such as CD45−, EpCAM+, CK 8+, and 19+, among others, in whole blood [24].
The detection of CTCs is closely associated with a diminishment of the OS in patients that are being treated for metastatic breast, prostate or colorectal cancer.
As this technique would not give a “complete” landscape of the tumor dynamics and patient monitoring, it should be performed and analyzed jointly with other clinical information derived from other types of diagnostic tests, such as ctDNA genetic tumor study, imaging, and the patient’s medical history, in order to give a proper management of the prognosis of the cancer.
Hayes and colleagues have utilized CellSearch for the detection of metastatic breast cancer and reported that a baseline of 5 CTCs in 7.5 mL of blood would be associated with poor OS among the 177 patients evaluated in the study, further reviewed in [25].
However, there are several cases in which cancer patients would lack EpCAM expression; hence, a different antibody, or even isolation method, would be necessary in order to capture CTCs in circulation [26].
There have been multiple developed technologies using several types of capture devices. Among them, there are microfluidic-based platforms, which would allow a better control of small volumes of reagents during several processes.
In 2007, Nagrath et al. [27] reported that CTCs, from whole blood, could be detected and captured using a microfluidic device based on an anti-EPCAM approach. Compared to previously reported studies, this approach isolated CTCs with better levels of enrichment and sensitivity.
Currently, several microfluidic platforms have used a combination of immunoaffinity- and size-based approaches in order to develop an integrated system capable of improving the purity and recovery levels of CTCs. For instance, Ozkumur et al. [28] described a microfluidic-based chip for CTC separation, known as iChip, which combines magnetic separation, hydrodynamic sorting, and inertial focusing of CTCs from blood.
Physical Properties
The key advantage of physical properties is that they allow a label-free separation of CTCs. These biomarkers have shown particular physical properties, including heterogeneity, buoyant densities and larger size (7–30 µm). Despite their evident heterogeneity, several CTC isolation platforms have been proposed for their capture from blood with considerably higher purity and recovery rates. Some of the main CTC-enrichment methods that are based on the physical properties include inertial sorting, density gradient separation, dielectrophoresis isolation, size-based filtration, and photoacoustic flow cytometer, among others [23].
Density Gradient Separation
In most studies, blood samples are diluted, put as a “coat” over the media and finally centrifuged in order to allow density-based separation. Some of these methods include Ficoll-Hypaquemedia, as well as the OncoQuick setup from Pharmacia-Fine Chemicals and Greiner BioOne, respectively. The last is supposedly better due to an incorporation of a porous membrane to decrease the number of contaminating blood cells with similar densities to CTCs.
Size-Based Filtration
This type of platform is used for the enrichment of CTCs based on their slightly larger size, compared to other hematopoietic cells. For instance, size-based isolation of epithelial tumor cells was employed to enrich CTCs from blood samples employing a polycarbonate-based membrane filter with cylindrical pores of 8 µm, approx. [29]. Nevertheless, cell fixation potentially limits subsequent genomic analyses. An alternative to this platform is the microfluidic approach involving microtraps, which leverage the size and deformability of CTCs, such as CytoQuest™ from Abnova Diagnostics (Taipei, Taiwan). Using size-based filtration methods also has several drawbacks, including relatively low flow rates and non-specific capture of cell debris.
Inertial Sorting
This approach employs hydrodynamic forces, which focus on blood cells at distinct streamlines in fluid. Sollier et al. has developed a size-based separation method using a combination of inertial focusing and microfluidic vortices. The basis of this approach consists of the use of inertia and fluid flow effects in order to separate different particles within a channel [30]. However, some major drawbacks are the possibility to isolate different blood cells (not the target) of similar sizes and a loss in detection if the CTC size is not within the working size range.
Altogether, with the recent advances in engineering and technology, several next-generation microfluidics platforms have been produced in order to recover CTCs with higher levels of purity and the potential to be cost-effective in the clinical setting.
Diagnosis and Recurrence Monitoring for Therapies
At present, the clinical value of CTC analysis remains controversial, even though there is proofing evidence indicating both a prognostic value and a correlation between the abundance of tumor cells in the blood of cancer patients and the response to therapy and, thus, treatment outcomes and OS.
Prostate Cancer
Prostate cancer presents particular complications in both patient monitoring and drug development. This peculiarity is based on two important aspects: (1) The fact that standard imaging approaches employed for the evaluation of the disease in bone, which is the most common location for cancer spread, have not been standardized yet; (2) The prostate-specific antigen (PSA) levels, which is the most common biomarker in the disease, might not reflect an accurate status of the disease.
An important finding for prostate cancer was that the second aspect aforementioned was verified. According to several studies, the correlation between the number of isolated cells and the overall disease status (based on PSA levels and disease spreading in bone) was discovered to be low. This reflected that the number of isolated cells represent an intrinsic property of the tumor. Based on several studies [31,32], patients presenting less than 5 CTCs in 7.5 mL of blood can be considered favorable and have a better prognosis than patients with more than 5 CTCs. Based on this, patient-specific treatments can be carried out in order to monitor and diminish the abundance of tumor cells for a better outcome.
Breast Cancer
Based on the WHO and several cancer-related studies, breast cancer is the most widespread neoplasm, with 5.2 million cases worldwide. Hence, there is an urgent need for improvements in the disease diagnosis and its patient-specific management. The majority of the information established on the CTC’s clinical setting is derived from several studies using a technology called CellSearch, which has demonstrated its importance in the prognosis of patients with numerous types of solid tumors, including prostate, colon, and breast cancer. Based on those results, a CTC cut-off level of ≥5 cells per 7.5 mL of blood was identified and correlated with a higher risk for disease progression and a considerably low OS [17].
Pancreatic Cancer
Based on their phenotype, the great majority of solid neoplasms formed in the pancreas are pancreatic ductal adenocarcinomas (PDACs). PDAC is diagnosed in most patients at an advanced stage, making it extremely difficult to foresee a good prognosis. Due to the fact that these tumors are mostly aggressive, and, according to several studies, they have a considerably poor response to cytotoxicity-based and targeted therapies, only 7% of patients survive for approx. 5 years [23]. One of the main challenges of PDACs is that the symptoms appear after the cancer has reached its threshold of manifesting by itself; therefore, is quite complicated to detect at an early stage. Several studies have suggested that the use of different technologies, such as CellSearch and CTC-Chip, based on the detection of CTCs in blood can contribute to an early detection. Allard and colleagues carried out a study using CellSearch in healthy subjects, and patients with a variety of metastatic carcinomas, including PDAC. Based on the CTCs counts, PDAC was the most complicated carcinomas to detect, presenting the lowest numbers of CTCs (mean, 2 ± 6 cells in 7.5 mL of whole blood) compared with prostate, ovarian, breast, and lung, among others [33]. However, they concluded that CTCs are extremely rare in healthy subjects but present in various metastatic carcinomas, so the monitoring of CTCs in blood before any symptoms may help in the early diagnosis of these diseases.
More information: B.R. McDonald el al., “Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer,” Science Translational Medicine (2019). stm.sciencemag.org/lookup/doi/ … scitranslmed.aax7392
Journal information: Science Translational Medicine
Provided by The Translational Genomics Research Institute
