What Are Clinically Relevant Levels of Cellular and Biomolecular

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What are Clinically-Relevant Levels of Cellular and Biomolecular Analytes? Shana O. Kelley ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00691 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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What are Clinically-Relevant Levels of Cellular and Biomolecular Analytes? Shana O. Kelley1,2,3,4*

1

Department of Chemistry, Faculty of Arts and Sciences, University of Toronto, Department of Pharmaceutical Science, Leslie Dan Faculty of Pharmacy, University of Toronto, 3Institute for Biomaterials and Biomedical Engineering, University of Toronto, 4 Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3M2

2

*Correspondence should be addressed to S.O.K. ([email protected])

Abstract: The ultimate goal of developing sensors for biomolecular analytes is to offer new tools for the analysis of clinical specimens for biomarkers of disease. It is thus important to understand the types of samples that are routinely used in the clinic for specific indications, and what the typical levels of relevant analytes are in these specimens. This Sensor Issues article summarizes information concerning levels of target molecules and cells that are of interest for the development of new diagnostics for infectious disease and cancer. Having this information in hand helps better define the “needle-in-a-haystack” challenge associated with developing robust sensors with the needed levels of sensitivity and specificity.

Keywords: Bioanalysis; DNA detection; protein detection; clinical samples, infectious disease; cancer

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The use of molecular-level information in disease diagnosis and management is becoming more routine and essential for the use of new tailored therapeutics.1 The development of new sensing strategies for disease-related nucleic acids, protein biomarkers, and rare cells is an important focus to enable diagnostic testing to become more decentralized and cost-effective. The sensitivity and specificity levels required for compatibility with clinical specimens, however, present a challenge that must be taken into consideration in order for new solutions to have relevance to medical applications. The sensitivity and specificity requirements for new biomolecular or cellular sensors are highly dependent on the type of application being pursued, and the relevant sample type. Levels of pathogenic organisms relevant to infectious disease can vary significantly according to sample type (swab, blood, urine, etc), and the type of molecular target (mRNA, human genomic DNA, viral genomic RNA/DNA, protein) being detected is also an important consideration.

Figure 1. Summary of concentrations of biomolecular analytes in clinical samples. Concentrations are shown in terms of cells or viral particles per milliliter of sample (bottom axis), and also in terms of the molarity of molecules that may be used as specific biomarkers (top axis, gray bars). Given that different analytes may be present with 1 3 different copy numbers ranging from 10s to 1000s, sensitivity requirements are relaxed by ~10 – 10 . Applications of bacterial detection are outlined in green, viral targets are outlined in purple (with a dotted line for indications where quantitative monitoring is required), and cancer biomarkers are shown in blue (circulating tumor cells), red (protein biomarkers), and orange (circulating nucleic acids).

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ACS Sensors, along with other journals focused on the development of new analytical tools, features many developments related to the detection of markers of disease. The detection of molecular targets associated with viral2 and bacterial pathogens3 is a major focus for sensor developers, and cancer-related targets including serum protein biomarkers,4 circulating nucleic acids,5 and circulating cancer cells6 are also commonly targeted with new sensing systems. The relevant sample types, molecular markers and sensitivity and specificity requirements are discussed in this article to highlight areas where challenges remain and new innovations will be needed to meet clinical requirements. The detection targets and levels present in clinical specimens are summarized in Figure 1, and types of samples and the indications discussed in this Sensor Issues article are summarized in Table 1 . Table 1. Types of samples and indications discussed

Type of clinical specimen

Relevant indications Bloodborne viruses

Blood

Bacterial infections Circulating tumor cells

Serum/plasma Urine

Circulating biomarkers (protein/DNA) Circulating biomarkers Bacterial infections Nasal bacterial colonization (nasal)

Swab

Viral respiratory infections (throat) Skin infections (skin wound)

Nasopharyngeal aspirate

Sputum




Viral respiratory infections

Bacterial lung infections

PERFORMANCE REQUIREMENTS FOR SENSORS TARGETING INFECTIOUS PATHOGENS

The rapid and sensitive detection of infectious pathogens is an important capability that can limit the spread of disease, control outbreaks, and improve patient outcomes for patients with lifethreatening infections. The specific identification of the cause of an infection is critical for the control of antibiotic resistance rates and improved antibiotic stewardship. The analysis of bacterial and viral pathogens (and less commonly, fungal pathogens) can be performed with a variety of sample types, and different indications possess different requirements for qualitative versus quantitative monitoring. In general, DNA and RNA sequences are preferred targets for infectious disease testing, as they directly correspond to the presence of an active infection.

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Bloodborne viruses. The diagnosis and control of life-threatening viral infections like Hepatitis C (HCV) and the Human Immunodeficiency Virus (HIV) relies heavily on molecular-level, nucleic acids based detection of viral sequences.7 Both are blood-borne viruses, and therefore low levels of viral particles must be detected against a large background of normal blood cells. For both of these viral pathogens, treatment outcomes have been improved significantly by serial monitoring to assess treatment efficacy. HCV is a flavivirus containing a single copy of a single-stranded RNA genome that infects over 2% of the population worldwide.8 Infection with HCV can have serious complications, including liver disease that can be fatal if the infection is not managed effectively.7b HCV has seven major genotypes that are treated with different therapeutic regimens, thus it is important for new methods to analyze the specific RNA sequences contained within the genome of this virus.9 At diagnosis, levels of the virus in the blood are typically ~ 106 IUs (infectious units) per milliliter of blood. This translates to a femtomolar concentration of viral RNA. Monitoring lower viral levels, however, is quite important given that effectiveness of HCV therapeutics are typically monitored by the quantitation of HCV viral load, and it is desirable to monitor until RNA levels are “undetectable”. HIV is a lentivirus that contains two copies of a single-stranded RNA genome. Upon diagnosis, levels can approach 107 copies per milliliter of plasma.10 Just as in the example above, the efficacy of treatment is ideally monitored by quantitating viral RNA and observing levels to drop to very low concentrations.11 Thus, HIV detection strategies must be quantitative, highly sensitive and exhibit a large dynamic range. Another set of important bloodborne viral detection targets are those transmitted by mosquitos that cause tropical diseases including the Dengue virus and the Zika virus.12 Both are flaviviruses carrying one copy of a single-stranded RNA genome. Dengue viral RNA can be detected in the blood, saliva or urine at levels ranging from 103 - 106 copies per milliliter.13 Upon diagnosis, ZIka RNA can be detected in plasma or urine at levels ranging from 102-105.14 Interesting, levels in semen can 1000x higher,15 which explains the sexual transmission of this type of infection. Influenza virus. Influenza is also a flavivirus that has a variety of subtypes depending on the identity of proteins displayed on the exterior of the viral particles. Diagnosis of flu can be performed using a nasopharyngeal aspirate, a nasal swab, or a throat swab, and the levels of virus are highly dependent on the sample type.16 In the aspirate samples, loads are typically 106-109 viral particles/ml, while for throat swabs and nasal swabs levels can be as low as 103 viral particles/ml. The higher levels of influenza obtained with aspirate samples are accompanied by higher levels of normal bacterial flora, so while the sensitivity requirements for this sample type are less stringent, there is a greater need for specificity. Bloodborne bacteria. Detecting and identifying bacteria in the bloodstream of a patient is one of the more challenging applications for biomolecular or cellular sensors, as the presence of as few as 1 – 10 cfus (colony-forming units) of bacteria in a milliliter of blood is enough to cause a life-threatening infection.17 A variety of gram-positive and gram-negative bacteria can be found in the bloodstream of infected patients including E. coli, S. aureus, and Klebsiella, along with

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drug-resistant strains. If a ribosomal RNA is used as a target, which can be present at a copy number up to 103 or 104, this represents a subfemtomolar concentration of RNA present with a overwhelming excess of non-target RNA in the sample. This is an enormous challenge for direct detection approaches as well as enzymatic amplification methods, and therefore molecular-level analysis is typically performed after expansion of bacteria in culture. Given the delays associated with bacterial culture, this is an important area for future work. Every hour that a blood infection remains untreated with an appropriate antibiotic, the probability that a patient will survive is diminished by 8%.18 Thus, finding ways to push the sensitivity and specificity of new biomolecular sensors for this application is critical. Bacterial skin infections. It is important to detect and identify bacterial pathogens that underlie skin infections, as leaving them untreated can lead to serious complications including system infection. Collecting a swab from a skin wound typically yields a sample with 103 – 106 cfus/ml.19 Monitoring patients for colonization of bacteria at anatomical sites that are not open wounds, like the nasal passages, typically yields samples with much lower levels approaching 102 cfu/ml.20 This is an important application for bacterial detection, as nasal colonization when it involves antibiotic-resistant bacteria like methicillin-resistance Staphylococcus aureus is important to monitor within healthcare-associated settings as the presence of this bacteria in a healthy carrier can be dangerous to immunocompromised patients. Tuberculosis. Tuberculosis (TB) is another important diagnostic target, as it is affects millions worldwide and the percentage of drug-resistant infections is growing in parts of the world that do not have access to sophisticated testing facilities. TB is typically detected with sputum extracted from a patient and can be present at high levels ranging from 107 – 108 cells/ml.21 However, sputum is a difficult sample type of work with given its viscosity and this presents a major challenge for the implementation of molecular-level methods as an alternative to culture. Urinary tract infections. The rapid, on-demand detection of pathogens that cause UTIs, typically E. coli, S. saprophyticus, P. aeruginosa, and Klebsiella, is an critical capability because these common infections are becoming increasingly drug resistant.22 UTIs are the most common infections diagnosed in North America, and can be resistant to 2nd and 3rd line antibiotics, making them very problematic to treat and dangerous to patients. A urine sample is typically called positive if it contains > 105 cfu/ml of bacteria.22 Urine is one of the cleanest clinical specimens and contains minimal material that can interfere with a detection assay, and therefore the detection of UTI-associated bacteria requires high–sensitivity assays that can discriminate they type of pathogen present, but the level of specificity required is less stringent than needed with other types of sample containing higher background levels of non-target cells.


SAMPLES AND TARGETS RELEVANT FOR CANCER DIAGNOSIS AND MONITORING

Cancer is increasingly being understood and treated at the molecular level, and the clinical relevance of circulating biomarkers continues to be proven out. Proteins, nucleic acids sequences and even intact cells are relevant for the management of cancer. Protein biomarkers as well as circulating nucleic acids (cNAs) and tumor cells (CTCs) are important targets for the developments of methods for cancer monitoring. In addition, the specific detection of tumor-specific markers in biopsy samples is an increasingly important capability for

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the selection of molecularly-targeted therapies. Sensitivity and specificity requirements for assays targeting these markers are also quite stringent, as they exist at very low levels in clinical samples and are typically present in the presence of a large excess of related molecules. Protein biomarkers. Proteins present in the bloodstream represent ideal markers for noninvasive screening of patients for cancer.23 This type of target has historically been the most widely used. For example, the prostate-specific antigen (PSA) is a protein that has been monitored for decades as an early sign that prostate cancer could be developing in a patient. While this marker is falling out of favor as it is not specific for cancer and its use as a marker may lead to overtreatment,24 it is still a useful example of a target marker that can be targeted with biomolecular sensors. PSA is present at low ng/mL levels in healthy subjects;24 a 1 ng/mL concentration of this protein corresponds to a molar concentration of ~ 30 pM. This protein marker would be present along with thousands of other serum proteins at varied concentrations, and thus any molecular receptor used to specifically recognize PSA must be able to achieve a high level of specificity. Even higher levels of sensitivity may be required to monitor other protein biomarkers, and it has also been proposed that monitoring fM concentrations of PSA in patients whose prostates were surgically removed is an important means to monitor recurrence.25 Circulating nucleic acids. Tumors are known to shed nucleic acids into the bloodstream, and the ability to monitor these molecular targets provides a means to collect genotypic information about a tumor noninvasively.26 Circulating DNA and RNA sequences specific to tumors are very challenging to target, however, as they are present with a large background of wild-type molecules. A typical approach that circumvents this issue is the targeting of known mutations that are tumor-specific. Detecting these sequences specifically therefore requires very high levels of specificity, with single-base resolution. Circulating nucleic acids are present in blood at ng/mL levels,27 which depending on the length of a fragment, corresponds to a picomolar concentration. But tumor-specific sequences constitute a very small percentage of the overall pool, thus femtomolar sensitivity is needed at a minimum. Circulating tumor cells. The analysis of intact cells released by tumors into the bloodstream presents another promising means to profile tumors noninvasively.28 This is another rare analyte – present at levels around 10 cells per milliliter of blood – and one that is massively outnumbered by normal blood cells of which ~ a billion are present in 1 mL. Even if a mutlicopy marker is being used as a target for CTC analysis, this dictates that only sub-attomolar concentrations are present in clinical samples. CTCs are therefore routinely captured and concentrated prior to analysis. A unique challenge related to CTC capture relates to the fact that these cells represent a moving target.29 As intact, living cells, their properties are dynamic as they circulate in the bloodstream. Some CTCs are more epithelial and can be targeted with recognition agents specific to epithelial factors, but others seem to lose their epithelial character once the enter the bloodstream and become more mesenchymal. Thus, in addition to the sensitivity and specificity challenges related to the

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analysis of these rare cells, dynamic changes in these cells must also be taken into consideration.


SUMMARY

Building effective sensors for biomolecular analytes of clinical relevance requires attaining high levels of sensitivity and specificity, which must be proven out with highly heterogeneous samples that are representative of clinical specimens. For some sample types – especially those encountered in infectious disease diagnosis - analyte concentrations can be at reasonable concentrations reaching femto- to picomolar levels, but the fact that biomolecular targets can be outnumbered by a million-fold excess of non-target species makes clinical specimen analysis challenging. Moreover, many applications require a high degree of precision for quantitative monitoring, making dynamic range as important as sensitivity and specificity levels. Many challenges remain where robust, reproducible biomolecular sensors will make an impact on important clinical problems, and this motivates significant continued effort in this area.


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24. Bruinsma, S. M.; Bangma, C. H.; Carroll, P. R.; Leapman, M. S.; Rannikko, A.; Petrides, N.; Weerakoon, M.; Bokhorst, L. P.; Roobol, M. J.; Movember, G. A. P. c., Active Surveillance for Prostate Cancer: A Narrative Review of Clinical Guidelines. Nat. Rev. Urol. 2016, 13, 15167. 25. (a) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C., Single-Molecule Enzyme-Linked Immunosorbent Assay Detects Serum Proteins at Subfemtomolar Concentrations. Nat. Biotechnol. 2010, 28, 595-9; (b) Thaxton, C. S.; Elghanian, R.; Thomas, A. D.; Stoeva, S. I.; Lee, J. S.; Smith, N. D.; Schaeffer, A. J.; Klocker, H.; Horninger, W.; Bartsch, G.; Mirkin, C. A., Nanoparticle-Based Bio-Barcode Assay Redefines "Undetectable" PSA and Biochemical Recurrence after Radical Prostatectomy. Proc. Natl. Acad. Sci., U.S.A. 2009, 106, 18437-42. 26. (a) Leung, F.; Kulasingam, V.; Diamandis, E. P.; Hoon, D. S.; Kinzler, K.; Pantel, K.; AlixPanabieres, C., Circulating Tumor DNA as a Cancer Biomarker: Fact or Fiction? Clin. Chem. 2016, 62, 1054-60; (b) Schwarzenbach, H.; Hoon, D. S.; Pantel, K., Cell-Free Nucleic Acids as Biomarkers in Cancer Patients. Nat. Rev. Cancer 2011, 11, 426-37. 27. Tamkovich, S. N.; Bryzgunova, O. E.; Rykova, E. Y.; Permyakova, V. I.; Vlassov, V. V.; Laktionov, P. P., Circulating Nucleic Acids in Blood of Healthy Male and Female Donors. Clin. Chem. 2005, 51, 1317-9. 28. (a) Alix-Panabieres, C.; Pantel, K., Challenges in Circulating Tumour Cell Research. Nat. Rev. Cancer 2014, 14, 623-31; (b) Chaffer, C. L.; Weinberg, R. A., A Perspective on Cancer Cell Metastasis. Science 2011, 331, 1559-64; (c) Lang, J. M.; Casavant, B. P.; Beebe, D. J., Circulating Tumor Cells: Getting More from Less. Sci. Trans. Med. 2012, 4, 141ps13. 29. Green, B. J.; Saberi Safaei, T.; Mepham, A.; Labib, M.; Mohamadi, R. M.; Kelley, S. O., Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials. Angew. Chem. Intl. Ed. Engl. 2016, 55, 1252-65.

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