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Synthetic biomarkers: a new way of early cancer detection

Synthetic biomarkers: a new way of early cancer detection

  • Time of issue:2022-11-24
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(Summary description)Early detection of cancer while it is still localized can improve the response to medical intervention for patients with most cancer types. The success of screening tools such as cervical cytology in reducing mortality has sparked great interest in new methods for early detection (e.g., using noninvasive blood or biofluidic biomarkers). However, biomarkers generated from early lesions are limited by fundamental biological and transport barriers, such as short circulation times and dilution in the blood, requiring highly sensitive methods to detect very low signal levels. In addition, individual biomarkers often lack specificity, and they may be elevated in non-cancerous conditions or present in multiple cancers, which requires the identification of multiple combinations of analytes to assess disease.

Synthetic biomarkers: a new way of early cancer detection

(Summary description)Early detection of cancer while it is still localized can improve the response to medical intervention for patients with most cancer types. The success of screening tools such as cervical cytology in reducing mortality has sparked great interest in new methods for early detection (e.g., using noninvasive blood or biofluidic biomarkers). However, biomarkers generated from early lesions are limited by fundamental biological and transport barriers, such as short circulation times and dilution in the blood, requiring highly sensitive methods to detect very low signal levels. In addition, individual biomarkers often lack specificity, and they may be elevated in non-cancerous conditions or present in multiple cancers, which requires the identification of multiple combinations of analytes to assess disease.

  • Categories:Blogs
  • Author:AIVD
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  • Time of issue:2022-11-24 16:55
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Early detection of cancer while it is still localized can improve the response to medical intervention for patients with most cancer types. The success of screening tools such as cervical cytology in reducing mortality has sparked great interest in new methods for early detection (e.g., using noninvasive blood or biofluidic biomarkers). However, biomarkers generated from early lesions are limited by fundamental biological and transport barriers, such as short circulation times and dilution in the blood, requiring highly sensitive methods to detect very low signal levels. In addition, individual biomarkers often lack specificity, and they may be elevated in non-cancerous conditions or present in multiple cancers, which requires the identification of multiple combinations of analytes to assess disease.

 

A review entitled "Synthetic biomarkers: a twenty-first century path to early cancer detection" was published in the journal Nat Rev Cancer (IF: 60.7) by the group of Professor Gabriel A. Kwong of the Georgia Institute of Technology and Emory School of Medicine, USA. In this review, the authors discuss the rationale for the development of synthetic biomarkers based on biological fluids. It is investigated how these strategies use tumor dysregulation features to amplify detection signals, use tumor-selective activation to increase specificity, and use natural processing of body fluids (e.g., blood, urine, and proximal fluid) to facilitate detection. Finally, the challenges that exist in preclinical development and clinical translation of synthetic biomarker diagnostics are highlighted.

 

These lessons provide the basis for emerging diagnostics based on bioengineered sensors (e.g., molecular probes or genetically encoded vectors) designed to take advantage of the dysregulated characteristics of early-stage tumors or their precursors to generate an amplified signal, and these exogenous sensors use tumor-dependent activation mechanisms, such as enzyme amplification, to drive the production and amplification of synthetic biomarkers. Cancer can also be detected by imaging systems that may have the essential features of synthetic biomarker approaches, such as reporter gene imaging. These emerging technologies are driving the development of early cancer detection, and synthetic biomarkers may be the future of early cancer detection methods.

 

Challenges in early cancer detection Early cancer detection is not universally positive for biomarkers that are consistently shed, such as proteins, from patient tumors, and secretion rates may vary by as much as four orders of magnitude even for cells of the same tumor type. In addition, biomarkers released by dead cells are shed only once, and their detection can be easily confused with background shedding from healthy tissue. For example, cell-free DNA (cfDNA) is released from non-cancerous cells throughout the body, making the somatic mutation ratio or variant allele frequency (VAF) of malignant versus normal cells increasingly difficult to detect at low tumor loads. Image

 

Analysis from the Treatment Tracker Non-Small Cell Lung Cancer Progression Study predicted that a primary tumor load of 1, 10, or 100 cm3 would result in a mean plasma VAF of 0.006%, 0.1%, or 1.3%, respectively. For a typical 4 ml of plasma, it is estimated that only an average of 6 molecules per tube carry the corresponding somatic mutation. Further exacerbating the technical challenge is the fact that biomarkers are diluted by large amounts of blood, resulting in degradation or removal in the circulation; for example, the circulating half-life of ctDNA in blood is less than 1.5 hours.

 

Although mathematical model-based predictions and genomic timeline studies consistently estimate a window of opportunity of at least ten years for early cancer detection, However, rapidly growing and highly aggressive cancers may progress rapidly within a relatively narrow window of months to years and are associated with poorer clinical outcomes. Examples include triple-negative breast cancer and highly plasmacytotic ovarian cancer (HGSOC) with tumors that have BRCA1 or BRCA2 mutations or homologous recombination defects. Advances in the field of synthetic biomarker research aim to address these challenges, with the main approach being early detection using activity-based or genetically encoded mechanisms. Synthetic biomarkers based on active molecules have a long clinical history of systemic administration with exogenous drugs to assess biological function in vivo. Activity-based synthetic biomarkers are based on this model and include sensors and small molecule probes activated by enzymes in the tumor or its microenvironment to provide a molecular amplification mechanism for tumor biomarkers. Protease-activated synthetic biomarkers Protease-activated synthetic biomarkers include peptide substrates bound to the surface of an inert vector that releases a reporter into the blood or urine for detection after being cleaved by tumor proteases. In addition to molecular signal amplification, another key strategy for reaching the desired limit of detection (LOD) for early detection is to use human physiological features to increase the concentration of synthetic biomarkers in biological fluids. One approach is to exploit the size filtration of the kidney by selecting carriers with a hydrodynamic radius greater than the glomerular filtration barrier (~5 nm) to prevent surface-bound peptides from being removed into the urine. Another key strategy is to enhance passive delivery to the tumor site. For example, the use of polyethylene glycol (PEG) polymers plus iron oxide nanoparticles (IONPs) has a higher passive diffusion rate and can increase delivery to tumors. Another approach is to functionalize the sensor using tumor-penetrating ligands that are involved in active transport pathways to the tumor microenvironment. Proteases are a mixture of enzymes capable of cleaving multiple substrate sequences, which limits the specificity of the detection of individual sensors. Therefore, another key design principle is to design a multi-sensor library to detect cancer through profiling. This approach requires that each synthetic biomarker in the sensor library be labeled with a unique molecule. Small molecule probes Given the increasing number of tumor-specific antigens, cell surface markers, and metabolic pathways that can be targeted as small molecules, some research has focused on engineering molecular probes to generate synthetic biomarkers for cancer detection.

 

Stable isotope-labeled small molecules have been widely used as diagnostic probes in research laboratories for more than 30 years. The advantages of stable isotope labeling include no radiation risk to patients, no difference in metabolism compared to unmodified counterparts, and high signal-to-noise ratios. The FDA has approved several isotope-labeled probes, including 13C-methacetin and 13C-cholate, which measure hepatic cytochrome P450 activity and hepatic shunt, respectively, in the context of hepatic fibrosis and liver dysfunction, and liver fibrosis is an important risk factor for hepatocellular carcinoma.

 

Natural volatile organic compounds (VOCs) present in patient breath samples have also been used in cancer diagnosis. Lang et al. used an isotopically labeled synthetic VOC called "D5 ethyl--D-glucuronide" (EtGlu), which is a deuterated metabolite of ethanol. After intravenous injection, EtGlu is enzymatically converted to D5 ethanol by β-glucuronidase, an extracellular enzyme secreted by solid tumors, and then detected from the breath by gas chromatography coupled with high resolution mass spectrometry. Genetically encoded synthetic biomarkers Advances in mammalian synthetic biology are driving the development of biosensing. In addition to activity-based probes, genetically encoded structures form another major group of strategies that use engineered components or cells to amplify the release of synthetic biomarkers. These approaches focus on strategies that drive the production or secretion of bio-orthologous reporters by resident or infiltrating cells in the tumor microenvironment. The main advantage of these approaches is the ability to transduce the production of synthetic biomarkers into cells of a specific phenotype, thus potentially reducing the number of false positives caused by background production in healthy tissue. Currently, there are three major classes of genetically encoded systems used to generate synthetic biomarkers, including vector-based systems, mammalian cell-based systems, and bacterial cell-based systems.

 

Vector-based systems rely on two key components: a tissue-selective or cancer-selective promoter to drive transcription and a synthetic biomarker designed to be secreted into blood or urine for detection. Tissue-selective promoters provide the first level of specificity, such as the promoter of the normally silent human telomerase reverse transcriptase (TERT), which encodes telomerase, an enzyme often activated in cancer cells for proliferation immortality, one of the hallmarks of cancer. Since TERT is expressed at high levels in about 90% of human cancers but is silenced in almost all somatic cells, the TERT promoter has been used to drive gene expression in a variety of tumor cells. A second component of the vector-based strategy is the secreted reporter, which serves as a synthetic biomarker and can be detected in blood or urine. One of the first reporters designed for in vivo application was secretory embryonic alkaline phosphatase (SEAP), an engineered form of human placental alkaline phosphatase that contains a stop codon in the membrane-anchored structural domain, converting it into a truncated but fully active secretory reporter. In xenograft tumor models, SEAP levels correlate directly with tumor size and cell number. Another commonly used reporter is luciferase. Synthetic mammalian cell-based biomarkers The recent clinical success of pericyte therapy has inspired attempts to engineer mammalian cells as living biosensors. A distinct advantage of cells as diagnostic vectors is that some cells are able to localize and infiltrate cancer sites compared to molecular probes, which are limited by their dependence on passive diffusion of the vascular system to accumulate in the tumor. Mesenchymal stem cells (MSCs) are adult pluripotent stem cells with regenerative and immunomodulatory properties, and Liu et al. used a mouse model to demonstrate that engineered MSCs can be used to detect cancer metastasis in the blood. First, MSCs were designed to secrete humanized glutamate, and after intravenous injection, engineered MSCs lasted longer in mice with lung metastases from breast cancer compared to tumor-free mice, resulting in higher blood levels of humanized glutamate. However, since MSCs exhibit chemotaxis to sites of inflammation and injury or may themselves be involved in cancer progression, more studies are needed to understand these potential limitations. Aalipour et al. further developed the concept of cell-based diagnostics using engineered macrophages as live cell sensors. It was found that tumor-associated macrophage M2-type reprogramming resulted in significant changes in arginase 1 (encoded by ARG1) levels and that macrophages that metastasize in solid tumors can upregulate arginase 1 up to 200-fold. Based on this finding, they used the ARG1 promoter to drive glutamate production during macrophage M2 polarization. This study lays the foundation for the concept of cellular immunodiagnosis, an approach that could also be extended to T cells, B cells, and natural killer cells, considering that many other immune cells similarly regulate metabolic gene expression in the tumor microenvironment. Bacteria-based synthetic biomarkers Certain types of bacteria infiltrate and selectively grow in tumors, which is attributed to increased levels of nutrients that inhibit immune surveillance and release from necrotic cells within the core of solid tumors. This has led to the use of engineered tumor-targeting bacteria as programmable vectors for cancer detection, and Panteli et al. have genetically modified an attenuated strain of Salmonella enterica that is 10,000 times less virulent than wild-type strains to release ZsGreen as a fluorescent biomarker or "fluorescent marker." After intravenous administration to tumor-bearing mice, the level of fluorescent markers in the serum depends on the tumor mass and can be mathematically modeled to predict its ability to detect tumors. Several challenges remain to be addressed in the use of bacteria for early cancer detection. Although engineered strains including Clostridium, E. coli, and Salmonella have been shown to be nonpathogenic to animals and humans, the inherent toxicity of bacterial components and the potential for reversion to toxicity pose safety concerns. In addition, it is unclear whether all tumor types and de novo lesions lacking a necrotic core can be colonized by systemically delivered bacteria. Advances in synthetic biology could provide solutions to these challenges while also providing opportunities to design "smart" microbes with specific and controlled behaviors. For example, bacteria designed using population-sensing biological circuits could be used for bacterial communication to synchronize activities and generate emergency behaviors, such as the timed release of therapeutic drugs to kill tumors or promote systemic anti-tumor immunity after a threshold population density is reached. Applied to the field of early cancer detection, these biological circuits have the potential to increase specificity by reducing background activity in healthy tissue. In the future, these genetically programmable vectors may have the potential to be developed into safe and regularly ingested foods (e.g., yogurt) for routine cancer screening or cancer chemoprevention. Preclinical studies of synthetic biomarkers have been reported in numerous preclinical studies, showing the potential of activity-based synthetic biomarkers to achieve the required LOD for early detection.

 

In a xenograft mouse model, an activity-based sensor consisting of IONPs combined with a unique peptide substrate was able to identify LS174T colorectal tumors in a volume 60% smaller than that detected by the serum biomarker CEA. Kwon et al. reported an activity-based molecule sensor that combined a tumor-penetrating peptide to target and increase its delivery to metastatic in situ ovarian cancer models. By quantifying synthetic biomarkers enriched in urine, they reported the ability to detect tumor metastasis with near-perfect accuracy (AUROC of 0.99) when the median node diameter was less than 2 mm and the mean total tumor load was 36 mm3. In contrast, human epithelial protein 4 (HE4) serum biomarkers were indicative of tumor metastasis only when the mean tumor load reached 88 mm3.

 

In vivo LOD studies on genetically encoded synthetic biomarkers have also been reported.  A study by Aalipour et al. showed that the engineered macrophage sensor for permetastasis detected medium-sized CT26 colorectal tumors (50–250 mm3 in volume) with 100% sensitivity and specificity, after comparing the performance of their macrophage sensor with In benchmark studies comparing the performance of LS174T tumor-secreted plasma CEA or CT26 tumor-released cfDNA, they reported a lower LOD. cEA detected tumors with a volume of approximately 136 mm3, whereas cfDNA detected tumors with a volume greater than 1500 mm3. Clinical Studies of Synthetic Biomarkers Human applications of synthetic biomarkers have not yet entered pivotal trials, as the field is still in its infancy. Currently, the most advanced synthetic biomarker in clinical trials is polyglycolylated peptide, which is well tolerated and safe in healthy volunteers based on preliminary data from a recent phase I study. The first few clinical use cases for synthetic biomarkers need to be carefully considered, as early failures could set the field back. Screening asymptomatic patients for early-stage cancer is a challenging endeavor and may pose ethical challenges in clinical trial validation studies. For example, patients who test positive for synthetic biomarkers may need to wait for imaging confirmation (i.e., allowing tumor growth) before therapeutic intervention can occur. Potential clinical entry points, such as pharmacodynamic assessment of treatment response or monitoring of recurrence after initial resection, could test the utility of synthetic biomarker approaches. Notably, as the field moves toward human trials, many of the components and vectors that comprise synthetic biomarkers are undergoing clinical evaluation, have a proven safety record in humans, or have received FDA approval. An example is the protease-activated substrate used in imaging probes for intraoperative detection of tumor margins. Similarly, for genetically encoded synthetic biomarkers, many clinical trials have highlighted the safety and utility of attenuated bacteria as vectors for targeting tumors and delivering therapies. These precedents provide a broader understanding of synthetic biomarker embodiments that will be safe and well tolerated in humans. Outlook: Although the emerging field of synthetic biomarkers is exciting and promising, there are gaps in our current understanding of cancer pathogenesis that need to be filled while addressing technical challenges to guide future progress. In particular, there is limited understanding of the biology of early lesions and when and how precursor lesions transform into malignancies, but this information is needed to guide sensor engineering strategies. This highlights the challenges in the field of synthetic biomarkers for early detection of cancer. In addition, there are many questions that need to be answered. For example, for which early-stage tumors or precancerous lesions can sensor engineering be used? How can machine learning support the identification of key features in complex biological datasets to achieve the predictive power needed for synthetic biomarkers? Which populations would benefit most from early detection? How often can patients be screened? Under what circumstances can the same probes be used to detect cancer recurrence? What is the cost of long-term surveillance for "high-risk" patients compared to the current standard of care? How can patient and tumor heterogeneity be overcome to ensure diagnostic accuracy? While there seem to be more unknowns than answers, we can be confident that through multidisciplinary efforts and the bold and innovative vision of scientists, solutions will emerge at an increasingly rapid pace.

 

Reference.

1. Synthetic biomarkers: the path to early cancer detection in the twenty-first century nat Rev Cancer. 2021 Oct;21(10):655-668.

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