The Application of Aptamers for Immunohistochemistry

Abstract

Immunohistochemistry has helped to make surgical pathology the “gold” standard for tumor diagnosis. However, given the numerous problems associated with the use of antibodies for the staining of cellular markers in paraffin-embedded tissues, there is a requirement for novel agents that have the advantages of antibodies, but with few of the disadvantages. Aptamers, which are chemical antibodies, are highly specific and sensitive, like their protein counterparts, but display few of the disadvantages. These molecules represent a unique reagent that has the potential to revolutionize the field of histopathological diagnostics. In this study, we present a review of some of the aptamers that have been validated for use in diagnoses and suggest some of the advantages to using these molecules in the future.

Introduction

Surgical pathology is considered the gold standard for tumor diagnosis [1 –4]. This process, involving the staining of tumor sections with Hematoxylin and Eosin (H&E), is invaluable for providing contrast between cellular components and allows the pathologist to detect microscopic morphological abnormalities to guide the staging and grading of the tumor for effective diagnosis [1,4]. However, when these morphological features alone are not adequate for conclusive diagnosis, such as in poorly or undifferentiated tumors, detection of cell-specific biomarkers through the use of immunohistochemistry (IHC) is routinely employed [1,2,5,6]. In the clinical pathology setting, IHC has diagnostic, prognostic, and therapeutic significance, which can directly impact upon patient management strategies and clinical decision making [7]. IHC works on the premise that each cell type has a characteristic marker, or epitope that can be used to differentially diagnose the cell of origin, thus supplementing the morphological characteristics observed with H&E staining [8,9].

Antibodies for IHC

For more than 40 years, antibodies have been routinely used in diverse clinical and research applications to selectively recognize specific epitopes within a target protein [4,5]. The visual detection of this antibody–antigen interaction, in most cases, requires conjugation of the primary (or a secondary) antibody to an enzyme, most commonly alkaline phosphatase or horseradish peroxidase. The reaction of these enzymes, following incubation with a chromogenic substrate such as DAB (3,3′-diaminobenzidine), results in a colored precipitate, which allows visualization of the presence and localization of the target molecule at the cellular level [5]. Antibodies may also be conjugated to fluorophores for use in immunofluorescence (IF) imaging, although this is not routinely performed in diagnostic laboratories due to issues surrounding photo bleaching, fluorescent quenching, and increased background staining. Fluorescence microscopy also requires the use of a dark field, which does not allow for simultaneous analysis of fluorescence patterns and cell morphology [5]. Furthermore, the fact that IF does not result in permanent staining, renders IF techniques impractical for routine tumor diagnosis [5]. Conversely, as chromogens are relatively light insensitive and chromogenic IHC techniques can be undertaken on an open bench with the results viewed using a simple light microscope [5], this technique is not only relatively straightforward to implement in a diagnostic laboratory, but also provides staining results in the context of overall cell morphology.

Both polyclonal and monoclonal antibodies are popular molecular recognition elements for use in IHC techniques, although monoclonal antibodies are preferred. Polyclonal antibodies are raised by injection of the target protein into various animal species, primarily rabbits. Since these antibodies are produced by different immune cells within the host animal, they will react with a variety of epitopes on the protein against which they were raised. This results in lack of specificity and significant batch-to-batch variation [4]. On the other hand, monoclonal antibodies are produced by an individual clone of hybridized plasma cells. However, to obtain commercially viable yields, monoclonal antibodies still generally require transplantation into the peritoneal cavity of animals for propagation. Since monoclonal antibodies are produced by a single clone of cells they will, in theory, recognize a single epitope within the target protein and are thus more specific than polyclonal antibodies [4]. These antibodies are capable of attaching to their target with high specificity and sensitivity, although the selection of the appropriate antibody is important. The specificity of the primary antibody is the most critical determining factor in the success of IHC techniques [5]. Indeed, a report has suggested that 42% of diagnostic inconsistencies are related to poor antibody selection and antibody crossreactivity [10,11]. Additionally, there are a number of other issues relating to the use of antibodies for IHC purposes.

Quality Concerns Over Commercially Available Antibodies

Inconsistency in antibody purity and specificity has been implicated, at least in part, in the lack of reproducibility in 47 out of 53 landmark preclinical cancer trials controversially reported by Begley and Ellis in 2012 [12,13]. There are currently more than 2 million commercially available antibodies, marketed by over 300 different companies [13]. Some variance in quality is perhaps not unexpected between manufacturers. This was assessed by Berglund et al., who performed standardized validation tests—including IHC on tissue microarrays and western blotting—to assess the quality of 1,410 monoclonal antibodies and 1,316 polyclonal antibodies from 51 different providers. The result was an average success rate of 49% for the detection of the target protein [14]. Although Berglund et al. admit that the success rates would have been higher if the assays were individually optimized for each antibody, their results also revealed considerable variability in product quality between suppliers. These inconsistencies were exhibited either by differences in reported staining patterns or by the existence of crossreactivity.

Additional issues surround the use of antibodies for IHC. One major problem is the nonspecific staining, which may result from ionic and hydrophobic interactions during antigen–antibody binding. Also, the attraction of basic groups present in the collagen fibers to the Fc fragment of the antibody may result in nonspecific staining [15]. Moreover, it has been reported that half of all IHC failures result from improper antibody storage in laboratories. Therefore, the use of antibodies for IHC requires a clear understanding of antibody characteristics, antibody titration, and optimization. However, even with the aforementioned problems taken into consideration, problems still arise due to batch-to-batch variation leading to discrepancies in staining intensity [7].

Aptamers as an Alternative Molecular Probe for IHC

Currently, antibodies are the only commercially available and clinically validated molecular probe for immunohistochemical applications [16]. However, in the past 25 years a new class of biorecognition molecules has emerged with distinct advantages over their antibody counterparts. Aptamers are short, single-stranded DNA or RNA oligonucleotides that are capable of binding to their target molecule with high specificity and affinity [2]. Aptamers form complex three-dimensional structures and bind tightly to their target through conformation-based interactions and are thus known alternatively as chemical antibodies [17]. Aptamers are generated by an in vitro process which was first described, concurrently, by three independent research groups [18 –20]. Tuerk and Gold [20] described the process of aptamer selection as the systematic evolution of ligands by exponential enrichment, thus the term SELEX still denotes the process today.

SELEX involves the incubation of a target of interest with a random pool of ∼10¹⁴ DNA or RNA oligonucleotides, typically between 30 and 50 base pairs (bp) in length [18,20]. Through iterative rounds of incubation with the target, nonbinding oligonucleotide species are removed, whereas bound species are amplified by polymerase chain reaction (requiring prior reverse transcription in the case of RNA) and returned to the next round of selection. Following ∼8–12 selection rounds, the resultant pool of ligands is enriched for species that are highly specific for the target [17]. The SELEX process is generally time consuming and takes several weeks to complete, but the result is one or more ligands with high specificity and affinity for their target antigen. Furthermore, the in vitro synthesis of aptamers virtually eliminates the issue of batch-to-batch variation [17,21]. A further advantage is that the target of interest is not limited to molecules which produce an immune response in the host animal, as for antibodies. Aptamers may be generated against virtually any target, including proteins, small molecules, whole cells, or even entire organisms [17,22,23]. Additional advantages of aptamers over their monoclonal antibody counterparts include flexible modifications and conjugation to therapeutic and diagnostic agents without loss of functionality, as well as their lack of toxicity and immunogenicity for use in in vivo applications [24]. Perhaps one of the most advantageous features of aptamers for both diagnostic and therapeutic applications is their relatively small size. The average aptamer (8–25 kDa) is around 10–15 times smaller than a monoclonal antibody (150 kDa) [25,26]. This confers considerable in vivo benefits, such as faster tissue diffusion and enhanced tumor penetration, which are advantageous for targeted drug delivery strategies [25]. While some optimization may still be required to optimize their pharmacokinetic profile, these aptamers still demonstrate more favorable kinetics than their antibody counterparts [27,28]. Potentially, there are also considerable benefits of this 10-fold reduction in size still to be realized for the purposes of IHC, particularly in applications for which epitope accessibility is reduced, such as in fixed tissues. This may also be relevant for the purposes of multiple histochemical staining, particularly when target biomarkers are colocalized within the same cellular compartment, and steric hindrance might be expected to compromise staining sensitivity [29].

Aptamer IHC

Over the past quarter of a century, technical improvements in the process of aptamer selection and advances in post-SELEX modification and optimization of aptamers have dramatically improved serum stability and functionality across a wide range of applications [30], making it possible to use aptamers for almost any application, which traditionally involves the use of antibodies [17]. However, the application of aptamers as molecular probes for immunohistochemical tissue analysis is still in its infancy. To date, only a limited number of publications have moved beyond fluorescence labeling and investigated the suitability of aptamers for the chromogenic staining of formalin-fixed tissues—the format preferred in routine histopathology [1] (Table 1).

Table 1.

A List of Aptamers Used in Immunohistochemical Applications

Aptamer name(s)	Aptamer target	Tissue fixation method	Immunostaining technique	Reference
SYL3C	EpCAM	FFPE and frozen tissues	Immunofluorescence: CY3-labeled secondary antibody	[36]
MMP2	MMP2	Frozen tissues	Chromogenic staining: HRP-DAB	[35]
DT3 and Ep23	EpCAM	FFPE	Chromogenic staining: HRP/DAB	[2]
			Immunofluorescence: DY647, TYE665, FITC (direct labeling)
1.5M short	Heparanase	FFPE tissue sections and formaldehyde-fixed cultured cell lines	Chromogenic staining: HRP/TMB, avidin/biotin/DAB	[26]
1.5M long
3.0M			Immunofluorescent staining: streptavidin/FITC
HER2 and EGFR SOMAmers	HER2; EGFR	Frozen tissues	Immunofluorescent staining: Direct Cy3 labeling	[6]
CD30 aptamer	CD30/TNFR family proteins	FFPE tissues	Chromogenic staining: HRP/DAB	[16]
III.1	Rat homologue of mouse pigpen	Frozen tissues	Immunofluorescence: direct FITC labeling	[31]
S1, S11e, S15 and S6	NSCLC adenocarcinoma cells	FFPE tissues	Immunofluorescence: direct TAMRA labeling	[32]
TSP-2, ESAM and MRC1 SOMAmers	TSP-2; ESAM; MRC1	Frozen tissues	Immunofluorescence: chromogenic staining: avidin–biotin–peroxidase	[33]

DAB, 3,3′-diaminobenzidine; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; ESAM, endothelial cell-specific adhesion molecule; FFPE, formalin-fixed paraffin-embedded; HER2, human epidermal growth factor receptor 2; HRP, horseradish peroxidase; MMP2, matrix metalloproteinase 2; MRC1, macrophage mannose receptor 1; NSCLC, nonsmall cell lung cancer; TAMRA, tetramethylrhodamine; TNFR, tumor necrosis factor receptor; TSP-2, thrombospondin-2.

Overall, IHC not only demonstrates the diagnostic potential of aptamers, but also represents a convenient and illustrative means for analysis of an aptamer's binding properties. IHC techniques have thus proved useful when multiple oligonucleotide sequences are generated by the SELEX process, as a means of assessing the specificity and sensitivity of aptamer candidates and selecting those with the most desirable, tumor-specific binding properties [2,26,31,32]. For example, Blank et al. were the first to demonstrate the use of IF staining for tumor analysis [31]. They selected DNA aptamers against transformed rat endothelial cells to selectively differentiate between normal vasculature and tumor microvasculature in a rat experimental model of glioblastoma. They employed IF techniques to analyze the 25 candidate oligonucleotide sequences they obtained to select for those which bound selectively to tumor microvessels. They reported that one FITC-labeled aptamer (III.1), which selectively bound the rat homologue of the mouse pigpen protein, selectively stained tumor microvessels of rat brain glioblastoma, but not the normal vasculature of peritumoral areas in frozen tissue sections.

In 2009, Zhao et al. [32] were the first to report the use of synthetic oligonucleotides as molecular probes for immunostaining of formalin-fixed paraffin-embedded (FFPE) tissues. They selected DNA aptamers against live cells of the adenocarcinoma subtype of nonsmall cell lung cancer (NSCLC). To test the ability of these aptamers to recognize certain NSCLC subtypes in clinical samples, they investigated the immunohistochemical staining of clinical FFPE samples of both tumor and matched normal lung tissues using aptamers fluorescently labeled with tetramethylrhodamine (TAMRA). They identified three different aptamer sequences, which selectively bound to membrane biomarkers of adenocarcinoma sections. Zhao et al. noted that nonspecific binding of DNA aptamers to cell nuclei may compromise specificity due to the presence of many nucleic acid-binding proteins in this cellular compartment. Therefore, they compared the staining patterns of the selected aptamers with the results obtained after incubation with a random, TAMRA-labeled DNA library in sections also counterstained with Hoechst 33258 to label cell nuclei. The random DNA library showed a nonspecific staining pattern, which overlapped with the nuclei counterstain. Conversely, the staining pattern obtained with the aptamers rarely overlapped with the Hoechst signal and was thus considered as specific for the cellular membrane.

In 2010, Zeng et al., [16] were the first to demonstrate chromogenic IHC techniques using aptamers and their results were compared directly with commercially available anti-CD30 antibodies. Using their anti-CD30 RNA aptamer, they demonstrated that the oligonucleotide showed no nonspecific or background staining in CD30-negative tissues and that its staining profiles in FFPE lymphoma sections were comparable or superior to sections stained with the standard CD30 antibody. Remarkably, the aptamer showed an optimized staining pattern at a reduced antigen retrieval temperature of 37°C and required an incubation time of only 20 min. In contrast, the CD30 antibody showed minimal to no staining under such conditions and required antigen retrieval at 96°C and an incubation time of 90 min to produce similar staining patterns. An additional benefit was the lack of aptamer staining observed in CD30-negative necrotic cells, a considerable problem observed with the CD30 antibody.

In 2011, Gupta et al. published an article identifying the nonspecific binding of DNA aptamers to cell nuclei as a significant barrier toward the successful use of aptamers for routine IHC [6]. They hypothesized that this was largely due to the electrostatic attraction of the aptamers to positively charged sites within the nuclei. To overcome this problem they attempted to generate aptamers with minimal ionic contributions to binding by conducting the SELEX selection in the presence of increasing dilutions of dextran sulfate (DS) throughout each iterative selection round. The purpose of this DS application was twofold, offering both a kinetic challenge and polyanionic competition. Two aptamers were generated in this fashion, the first against the epidermal growth factor receptor (EGFR) and the second against the human epidermal growth factor receptor 2 (HER2). These were termed slow off-rate modified aptamers, or SOMAmers, as a result of their high target affinity and slow dissociation rate. The SOMAmers were validated by IF in frozen tissues. In addition, the authors claimed that similar results were obtained with standard chromogenic methods. Specificity of the SOMAmers was assessed through the use of positive and negative control tissues, but unfortunately no antibodies were investigated to allow for direct comparison of sensitivity or specificity. Notably, in breast carcinoma tissue, the HER2 SOMAmer bound to cell membranes in the expected pattern only when coapplied with 1 mM DS. Nonspecific binding of the HER2 SOMAmer to nuclei, stroma, and cytoplasmic cell constituents in the absence of coincubation with DS was observed in every tissue sample investigated. Remarkably, the HER2 SOMAmer was found to have a fast association rate, causing saturation of the target in less than 1 min at 1 μM concentration and less than 5 min at 100 nM, suggesting that incubation times for intraoperative IHC procedures could, in some cases, be reduced to as little as a few minutes with the use of aptamers as diagnostic probes. However, Gupta et al. reported the failure of either SOMAmer to specifically stain for its respective biomarker in FFPE tissues.

In 2012, Mehan et al. [33] also described the use of DNA SOMAmers as histochemical probes for biomarkers of NSCLC. Mehan et al. applied a highly multiplexed proteomic assay (SOMAscan) to identify changes in protein expression profiles from tissue homogenates of NSCLC and matched healthy lung tissue. The 36 proteins, which exhibited the greatest difference in expression between healthy and diseased tissues, were highlighted—16 of which were novel markers for NSCLC. Of these, three were investigated as novel histochemical probes. Both chromogenic and IF staining techniques in frozen tissues were investigated with the thrombospondin-2 (TSP-2), endothelial cell-specific adhesion molecule (ESAM), and macrophage mannose receptor 1 (MRC1) SOMAmers. Histochemical staining results were again enhanced by the inclusion of DS during binding and washing steps and were congruent with the direction of change in protein expression between healthy and diseased tissues. Large-scale proteomic comparison was thus shown in this case to successfully identify novel histochemical probes. Conversely, histochemical analysis also provided valuable information on biomarker distribution and was thus helpful for validating the role of the identified biomarkers as disease-specific, rather than being attributed to related biochemical changes—such as those involved in inflammation.

Also in 2012, Simmons et al. [26] generated a DNA aptamer against heparanase—an extracellular matrix remodeling enzyme secreted by metastatic tumor cells. Chromogenic staining was performed in FFPE first-trimester and full-term placental tissues, with variable heparanase expression, to test the specificity of the aptamer. Heparanase staining with the aptamer was comparable and in some cases superior to the anti-heparanase antibody (HPSE1), demonstrating potential for this aptamer in future IHC applications.

In 2013, Shigdar et al. investigated the validity of two anti-EpCAM (epithelial cell adhesion molecule) aptamers (DT3 and Ep23) [34] in FFPE tissues [2]. The tissues tested included colon cancer (HT-29) xenograft sections, with high-level EpCAM expression, three breast cancer subtypes, with low to moderate EpCAM expression, and glioblastoma xenograft sections as negative controls. EpCAM expression in all cell lines was investigated quantitatively by western analysis and qualitatively using flow cytometry. The sensitivity and specificity of each aptamer was analyzed through both IF and chromogenic staining and the results were compared to those obtained with the EpCAM antibody 323/A3.

Shigdar et al. built upon the earlier observation by Gupta et al. [6] that anionic competition with DS inhibited nonspecific nuclear staining. It was hypothesized that, since aptamers are nucleic acid molecules, a combination of methods used in traditional IHC and in in situ hybridization may be more successful. They, therefore, incubated the aptamers in the presence of DS, which not only reduced nonspecific binding, but also accelerated the rate of nucleic acid hybridization, leading to reduced incubation times. The aptamers were also incubated in the presence of heparin, which was shown to reduce background staining. If either DS or heparin were omitted from the hybridization buffer, positive staining did not occur, even if incubation time was increased. With this method, markedly superior and sensitive IF and chromogenic staining was achieved with the aptamers in both breast and colon cancer tissue in 90% of the samples tested. Of note was the failure of the DT3 aptamer to stain the HT-29 cells that possessed a high-level EpCAM expression. This was attributed to the masking of the epitope in these tissues for which the DT3 aptamer is specific, a problem not confined only to aptamer IHC, but also commonly seen with antibodies. Both the Ep23 and DT3 aptamers are RNA oligonucleotides of 19 bp in total, however, the proposed binding loop of the DT3 aptamer is only 4 bp compared to 10 bp for Ep23. While DT3 stained well in breast cancer xenografts of varying EpCAM expression, superior sensitivity was obtained with the Ep23 aptamer, especially in the case of HT-29 xenografts. Additionally, when tested in breast cancer patient FFPE samples, the aptamer showed superior specific staining of EpCAM (Fig. 1) [2]. Furthermore, incubation time required for the Ep23 aptamer was just 15 min compared to overnight for the 323/A3 antibody.

FIG. 1.

Tissue immunostaining of breast cancer and lymph node metastasis by EpCAM antibody and aptamer. EpCAM antibody immunostaining was weaker in the breast tumor (A) ×40 and (B) ×400 in comparison to EpCAM aptamer immunostaining in patient FFPE samples (C) ×40 and (D) ×400; all pictures were taken under a light microscope with ×40 magnification and ×400 magnification (taken from the center of the ×40 magnification) [2]. EpCAM, epithelial cell adhesion molecule; FFPE, formalin-fixed paraffin-embedded. Color images available online at www.liebertpub.com/nat

In 2014, Han et al. [35] verified the applicability of their matrix metalloproteinase 2 (MMP2) aptamer to chromogenic immunostaining in frozen tissues. Han et al. were able to detect the MMP2 protein in tissues, including atherosclerotic plaque and gastric cancer samples, with results similar to those observed for the anti-MMP2 antibody.

Recently Pu et al. [36] were the first to compare the performance of an aptamer as a molecular probe in both frozen and FFPE tissue sections. Pu et al. demonstrated the efficacy of the SYL3C DNA aptamer against EpCAM [37] for the immunostaining of colorectal tumor sections. The aptamer was conjugated with the fluorescent label (CY3) for immunofluorescent examination. The specificity of the aptamer was demonstrated by a lack of crossreactivity with either benign or inflamed colon tissue. However, the sensitivity of the aptamer was not validated, in contrast to the studies described above, where binding assays were performed [2,6,26]. The aptamer showed IF staining profiles comparable to the two anti-EpCAM antibodies employed for comparison. The authors noted a considerable reduction in processing time when working with the directly labeled aptamer as it allowed for the omission of the secondary antibody incubation step required for staining with the traditional antibodies.

It is also worth noting that aptamers have also demonstrated effectiveness in both peroxidase- and fluorescence-based cytochemistry techniques. Although the terms IHC and immunocytochemistry (ICC) are often used interchangeably, there exist significant differences between the two. Unlike IHC, which involves sectioning of intact tissues, ICC involves fixation and analysis of suspended cells, which may be derived from either clinical samples or from laboratory culture. Therefore, in ICC most, if not all, of the extracellular components are removed, leaving a sample containing mainly separate, whole cells [38]. One exception to the dearth of information regarding aptamer cytochemistry in fixed cells is offered by Bruno et al. [39]. Bruno et al. demonstrated a novel approach for the detection of acetylcholine (ACh) using their ACh 6R DNA aptamer in Neuro-2a murine neuroblastoma cells induced to differentiate by treatment with 1 μM all-trans-retinoic acid for 5–7 days. For chromogenic staining, slides were first incubated with the biotinylated aptamer followed by incubation with a diluted streptavidin-peroxidase conjugate before color development with DAB. Fluorescence staining was performed directly with the FITC-labeled aptamer. The ACh was immobilized in situ by fixation with either cold 2% paraformaldehyde or cold alkaline allyl alcohol plus glutaraldehyde. With the aptamer–peroxidase method positive ACh staining was observed in 50%–75% of cells fixed with paraformaldehyde, but only 10%–25% of cells treated with allyl alcohol–glutaraldehyde fixation. Paraformaldehyde fixation also proved more successful in fluorescence techniques, in which allyl alcohol–glutaraldehyde fixation resulted in increased autofluorescence in the absence of the aptamer. This background fluorescence meant that aptamer-fluorescence staining was possible only with the 2% paraformaldehyde fixation method.

Aptamer Histochemistry in Formalin-Fixed Tissues

Aptamer-based IHC is a newly emerging field, with only a handful of example publications to be found among the 10,000 or so articles regarding aptamers currently in circulation [40]. To our knowledge, only three of these publications have investigated the use of chromogenic dyes for the staining of FFPE tissues—the format preferred in routine histopathology laboratories [2,16,26] (Table 1). This is in part due to the difficulties, for both aptamers and antibodies, in recognizing antigenic sites that have been masked or degraded by the process of formalin fixation [5]. Furthermore, optimization of antigen retrieval parameters is generally necessary and time consuming for successful staining results in FFPE tissues and often involves protease digestion or the boiling of sample slides in buffers of varying pH.

At least in the case of SOMAmers, there is admission of a failure to stain for respective biomarkers in FFPE tissues [6,33]. It should be noted that SOMAmers are described by Gupta et al. [6] as containing nucleotides with novel modifications designed to mediate hydrophobic interactions between the oligonucleotide and its target. Furthermore, there exists some evidence that aptamers may be more sensitive to subtle alterations in protein conformation [41]. Antibodies, in contrast, many times recognize a linear epitope. Therefore, the significant degree of protein denaturation generated by traditional antigen retrieval methods may be one reason for the improvements in staining intensity generally achieved with antibodies. Such antigen retrieval methods may, however, prove suboptimal for use with aptamers due to an increased reliance on the conformation-based recognition of secondary structures [16]. Thus, the development of novel methods of antigen retrieval in fixed tissues may be of benefit to the field of aptamer histochemistry. Another possibility to overcome the issues of altered protein conformation in fixed tissues is to raise aptamers intended expressly for diagnostic applications by performing the SELEX selection directly against the target molecule in its formalin-fixed conformation. This has been demonstrated previously by Li et al. by utilizing an in situ slide-based SELEX process to select an aptamer against hnRNP A1 in FFPE breast cancer biopsies, but has not yet been investigated for efficacy in IHC staining [42].

Clinical Significance of Aptamers for Use in IHC

Despite the limited published body of work regarding aptamer-based IHC, aptamers have consistently demonstrated promising results, often at greatly reduced incubation times. Thus, aptamers have the potential to streamline certain aspects of current intraoperative surgical pathology protocols. Currently, routine IHC procedures may require up to 24–48 h to complete, with antibody incubation representing the rate-limiting step [43]. Rapid IHC protocols have the potential to intraoperatively detect occult micrometastatic and microinvasive disease and, in doing so, may preclude the need for further surgeries [4,6]. Intraoperative IHC may also help to elucidate the source of tumors or metastases of unknown primary origin [44]. During neurosurgery, rapid IHC may allow for differentiation between operative and nonoperative neoplastic lesions and also guide the accurate identification of tumor margins [43]. Finally, aptamers can meet the requirement of the US Food and Drug Administration (FDA) for a companion diagnostic test as the same aptamer can be used for both diagnostic and therapeutic applications with no loss of functionality [2,28].

Conclusions

Being clinically validated and commercially available, antibodies have been used as a diagnostic tool for almost 40 years. Indeed, they have helped to revolutionize the field of diagnostics, where the identification of cellular markers can aid in diagnosis. However, there have been numerous reports of false negative and false positive results in procedures using antibodies, as well as the significant issues relating to batch-to-batch variation. Aptamers are chemically synthesized and thus, there is limited variation in their activity or structure recognition. Compared with antibodies, aptamers have proven to be more sensitive and produce less nonspecific background staining. Indeed, the results to date suggest that aptamers can provide a robust and cost-effective tool to translate biomarker discovery into pathological diagnostic practice to better stratify patients for personalized medicine. In addition, aptamers have the potential to speed up the diagnostic procedure and dispense with the requirement for secondary reagents. One recent editorial “Where are all the aptamers?” clearly described that only a few laboratories have utilized aptamers, and suggested that it may take another few years to establish aptamers in clinical applications [45]. The paragon to describe this is that the first antibody was produced in 1975, a single monoclonal antibody was approved by FDA in 1986, although it took a further 10 years to reach the market [46]. Currently, aptamers are in the development phase, from preclinical to clinical trials and even one therapeutic FDA approved aptamer in 2004. Great efforts have been taken to make use of aptamers instead of antibodies for utilizing them as both therapeutic and diagnostic agents. Given that aptamers have been shown to be more sensitive than their antibody counterparts, there is definite potential for the application of these molecular probes in future histopathological diagnosis and potentially for therapeutic applications.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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