Advances in HER2 testing

CHAPTER FOUR

Advances in HER2 testing Yun Chen*, Liang Liu, Ronghua Ni, Weixian Zhou School of Pharmacy, Nanjing Medical University, Nanjing, China *Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4.

Introduction Biological significance of HER2 HER2 amplification/overexpression in human breast cancer Commercial assays 4.1 IHC 4.2 FISH 4.3 Bright-field ISH 5. Methods in development and not yet approved by FDA 5.1 New methods for HER2 protein expression 5.2 New methods for HER2 gene detection 6. Detection of the HER2 protein in serum samples 6.1 ELISA 6.2 Surface plasmon resonance (SPR) 6.3 Electrochemical detection 6.4 QD-based detection 6.5 Mass spectrometry 7. Conclusions References

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Abstract HER2-positive breast cancer is a particularly aggressive type of breast cancer. Indication of HER2 positivity is essential for its treatment. In addition to a few FDA-approved methods such as immunohistochemical (IHC) detection of HER2 protein expression and in situ hybridization (ISH) assessment of HER2 gene amplification, several novel methods have been developed for HER2 testing in recent years. This chapter provides an overview of HER2 testing with emphasis on those new methods.

1. Introduction Breast cancer is the most common cancer among women worldwide, representing 30% of new cancer diagnoses. In addition, it is the second leading cause of cancer deaths among women [1]. With regard to the risk Advances in Clinical Chemistry, Volume 91 ISSN 0065-2423 https://doi.org/10.1016/bs.acc.2019.03.004

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Fig. 1 Kaplan-Meier survival curve for the prediction of disease-related death in lymph node-negative breast cancer by amplification of the HER-2/neu oncogene. Reprinted with permission J.S. Ross, J.A. Fletcher, The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy, Stem cells 16 (1998) 413–428.

of breast cancer, proper diagnosis and stratification of tumors for individualized precision therapy is of the utmost importance. There is mounting evidence that human epidermal growth factor receptor 2 (HER2) plays an important role in the occurrence and development of breast cancer and the following treatment [2]. HER2-positive (gene amplification or protein overexpression) breast cancer is a particularly aggressive type that includes a higher mortality at early-stage, reduced time to relapse, and an increased incidence of metastases [3,4] (Fig. 1). More importantly, its status may predict the response to chemotherapy and hormonal therapy. Indication of HER2 positivity is essential for treatment with anti-HER2 therapies, such as trastuzumab, pertuzumab and lapatinib, which have shown significant benefits in clinical practice [5,6]. For this reason, the testing of HER2 status is important for the management of breast cancer patients.

2. Biological significance of HER2 In 1985, King et al. found that DNA from human breast cancer had amplification of the HER2 gene [7], and 2 years later, Slamon et al. reported that this amplification is important in the pathogenesis and progression of breast cancer [8]. Since then, HER2 gene amplification and the resultant protein overexpression have been correlated with important breast tumor cell proliferation and survival pathways.

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HER2 is a 185-kd transmembrane tyrosine kinase receptor belonging to the family of epidermal growth factor receptors (EGFRs). The corresponding HER2 (ERBB2) oncogene is located on the long arm of chromosome 17q12. HER1 (EGFR), HER3 (erbB3), and HER4 (erbB4) are the other members in the EGFR family. All these tyrosine kinase receptors are single subunit transmembrane glycoproteins, which are composed of three domains, including the extracellular ligand-binding domain, the transmembrane domain, and the intracellular tyrosine kinase catalytic domain. Upon ligand activation, the receptors undergo conformational changes that allow their homodimerization or heterodimerization, followed by transphosphorylation, which activates several intracellular signaling pathways, such as the phosphatidylinositol 3 kinase/Akt (PI3K/ AKT) pathway, the Ras/mitogen-activated protein kinase (RAS/MAPK) pathway, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway, and the phospholipase C (PLC) pathway [9]. Transcription factors activated by these pathways regulate many genes involved in cell proliferation, survival, differentiation, motility, and adhesion. To date, there is no known ligand for HER2 receptors to form homodimers, whereas HER2 relies on heterodimerization with other family members or homodimerization with itself when expressed at very high levels [10]. In the EGFR family, HER2 has the strongest catalytic kinase activity, and HER2-containing heterodimers have the strongest signaling activities [11,12], thus playing a central role in those cellular processes. Therefore, aberrant expression of HER2 has been implicated in various cancers, especially in breast cancer.

3. HER2 amplification/overexpression in human breast cancer In general, normal breast tissue samples have shown no evidence of HER2 amplification and overexpression. However, in breast cancer tissues collected from patients, there are approximately 20% of breast cancers characterized by HER2 amplification/overexpression [13]. Among them, the HER2 gene may amplify 25–50 times and the HER2 protein may increase 40- to 100-fold [14]. As is well known, amplification of the HER2 gene is the major mechanism leading to protein overexpression, accounting for approximately 90% of such cases [15]. However, for a small proportion, protein overexpression may be due to transcriptional upregulation. Recent integrated

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proteogenomic research found that the protein expression may not be consistent with the level of gene or mRNA [16]. Currently, some believe that a protein used as a downstream regulator may be more reliable as a phenotype predictor. Much evidence has demonstrated that the differential HER2 level between normal tissue and a tumor helps to define HER2 as an ideal diagnostic and treatment target. Thus, HER2 status can be incorporated into a clinical decision, along with other prognostic factors, such as the estrogen receptor (ER) and progesterone receptor (PR), regarding whether to give any adjuvant therapy. In clinical practice, HER2-positive breast cancer is normally treated with the monoclonal antibody, trastuzumab. Trastuzumab, marketed by Roche as Herceptin®, the first drug that targets HER2, is a recombinant humanized murine monoclonal antibody that targets the extracellular domain of HER2, resulting in inhibition of tyrosine kinase activity and cellular proliferation in tumors caused by HER2 overexpression [17]. The addition of trastuzumab to the chemotherapy regimen has been associated with a longer time to disease progression, a longer duration of response, a higher 1 year survival rate, and longer overall survival [17]. First released in 2007, updated in 2013 and newly focused updated in 2018, the recommendations by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human HER2 testing Expert Panel are aimed at improving the analytic validity of HER2 testing and the clinical utility of HER2 as a predictive biomarker for potential responsiveness to therapies targeting the HER2 protein [18–20]. These guidelines pointed out that all newly diagnosed patients with breast cancer must have a HER2 test performed. Patients who then develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available [20]. The set of recommendations from 2007, 2013 and 2018, highlighting changes, is listed in Table 1. Upon the request of guidance, a few methods have been developed for the detection of HER2 status in breast cancer specimens in clinical practice at the levels of protein and DNA (Table 2). Those assays have been approved by the FDA, such as immunohistochemical (IHC) detection of HER2 protein expression, in situ hybridization (ISH) assessment of HER2 gene amplification like fluorescent ISH (FISH). Currently, these two assays are most commonly used. Both IHC and FISH have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features in tissue sections [39]. However, inconsistent results of IHC

Table 1 Summary of 2007, 2013 and 2018 HER2 test guidelines and recommendations. Topic

2007 Recommendations

2013 Recommendations

2018 Recommendations

Specimens to be tested

All primary breast cancer specimens and All newly diagnosed patients with breast cancer No change metastases should have at least one HER2 must have a HER2 test performed. Patients who then test performed develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available

Optimal See Table 3 algorithm for HER2 testing Test is rejected and repeated if: ISH rejection Test is rejected and repeated if: (1)–(5) are same as 2007 and report HER2 test criteria (1) Controls are not as expected; result as indeterminate as per parameters described (2) Observer cannot find and count at least two areas of invasive tumor; (3) 25% of signals are unscorable due to weak signals; (4) 10% of signals occur over cytoplasm; (5) Nuclear resolution is poor; (6) Autofluorescence is strong

No change

ISH Interpretation performed by counting at interpretation least 20 cells; a pathologist must confirm that counting involved invasive tumor criteria followed

The pathologist should scan the entire ISH slide before counting at least 20 cells or use IHC to define the areas of potential HER2 amplification If there is a second population of contiguous cells with increased HER2 signals/cell and this cell population consists of >10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and reported

The pathologist should scan the entire ISH slide before counting at least 20 cells or use IHC to define the areas of potential HER2 amplification If there is a second population of cells with increased HER2 signals/cell and this cell population consists of >10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and reported For brightfield ISH, counting requires comparison between patterns in normal breast and tumor cells because artifactual patterns may be seen that are difficult to interpret. If tumor cell pattern is neither normal nor clearly amplified, test should be submitted for expert opinion

Continued

Table 1 Summary of 2007, 2013 and 2018 HER2 test guidelines and recommendations.—cont’d Topic

2007 Recommendations

2013 Recommendations

2018 Recommendations

Acceptable (IHC and ISH) tests

Should preferentially use an FDA-approved IHC, bright-field ISH or FISH assay

No change

IHC rejection Test is rejected and repeated or tested by criteria FISH if: (1) Controls are not as expected; (2) Artifacts involve most of sample; (3) Sample has strong membrane staining of normal breast ducts (internal controls)

No change

No change

IHC Positive HER2 result requires interpretation homogeneous, dark circumferential criteria (chicken wire) pattern in 30% of invasive tumor. Interpreters have method to maintain consistency and competency

Should interpret IHC test using a threshold of more No change than 10% of tumor cells that must show homogeneous, dark circumferential (chicken wire) pattern to call result 3+, HER2 positive No change

Reporting requirements for all assay types

Report must include guideline-detailed elements

Report must include guideline-detailed elements except for changes to reporting requirement and algorithms defined in this table

Optimal initial test validation

Initial test validation requires 25–100 samples tested by alternative validated method in the same laboratory or by validated method in another laboratory Proof of initial testing validation in which positive and negative HER2 categories are 90% concordant with alternative validated method or same validated method for HER2

Laboratories performing these tests should be following No change all accreditation requirements, one of which is initial testing validation. The laboratory should ensure that initial validation conforms to the published 2010 ASCO/CAP recommendations for IHC testing of ER and PgR guideline validation requirements with 20 negative and 20 positive for FDA approved assays and 40 negative and 40 positive for LDTs. This requirement does not apply to assays that were

previously validated in conformance with the 2007 ASCO/CAP HER2 testing guideline and to those who are routinely participating in external proficiency testing for HER2 tests, such as the program offered by CAP Laboratories are responsible for ensuring the reliability and accuracy of their testing results, by compliance with accreditation and proficiency testing requirements for HER2 testing assays. Specific concordance requirements are not required Optimal internal QA procedures

(1) Ongoing quality control and equipment maintenance; (2) Initial and ongoing laboratory personnel training and competency assessment; (3) Use of standardized operating procedures including routine use of control materials; (4) Revalidation of procedure if changed; Ongoing competency assessment and education of pathologists

Should review and document external and internal No change controls with each test and each batch of tests; (1)–(4) are same as 2007; Should perform ongoing competency assessment and document the actions taken as a part of the laboratory record

Table 2 Main characteristics of the FDA approved assays. Method of Specimen analysis Trade name type Detection mechanism

Advantages

Limitations

References

IHC

FFPE HercepTes; PATHWAY; tissue InSite; Bond Oracle

Slides are incubated with an antibody directed against the HER2 protein, labeled, and finally the protein is made visible with a chromogen resulting in membrane staining

Results can be affect by [21–23] Easy to perform, relatively many pre-analytical cheap, less time consuming and no specialized equipment factors is required

FISH

INFORM; PathVysion; PharmDx

FFPE tissue

Fluorescent-labeled probe hybridization with target tumor DNA, and detected upon excitation of the fluorochromes and visualized using a fluorescent microscope

More reliable, sensitive, reproducible and accurate, pre-analytical factors have less impact on assay results, testing results is relatively straightforward

InstantQuality FISH

PharmDx

FFPE tissue

CISH

INFORM; SPoT-Light; PharmDx

FFPE tissue

More time-consuming and more expensive, requires costly equipment

[24,25]

Decreases the assay time, As same as FISH, only the hybridization buffer (IQFISH buffer) allows the determination in 1 day is different

As same as FISH

[26,27]

A peroxidase enzyme-labeled probe hybridization with target tumor DNA, and made visible with diaminobenzidine, and detected by bright-field microscope

[28–30] Technical problems, including tissue fixation time, tissue digestion time, and high background debris, can lead to erroneous results or loss of signal

Allows the simultaneous evaluation of tissue morphology and copy number alterations, tumor heterogeneity is promptly recognizable

Blood sample

ELISA

Immuno-1; ADVIA Centaur

Dual-ISH

INFORM FFPE HER2 Dual tissue ISH DNA probe cocktail

Foundation Next generation One CDx sequencing

FFPE tissue

Might not be reliable if [31–33] the serum samples are from patients receiving trastuzumab treatment

Using two monoclonal antibodies recognizing two distinct epitopes of the HER2 EDC. Then incubated with the second monoclonal antibody, which is labeled with horseradish peroxidase (HRP). After application of the HRP substrate, detection is accomplished by assessing the colored end product with spectrophotometry

A quick and simple assay in addition to being a less invasive (only blood samples are needed) and quantitative test, can be used to monitor the dynamic changes of HER2 status

After hybridization, HER2 probe is visualized with two antibody, then developed with a silver precipitate, and visualized as discrete black spots. Sequentially, DNP-labeled CEP17 probe is applied to the same slide, visualized with two antibody, then developed with a fast red reagent. The CEP17 signals are visualized as red spots

The same disadvantages [34–36] Allows the simultaneous as CISH visualization of both HER2 and CEP17 targets on the same slide, procedure is completely automated, reproducibility of results is increased, since risk of human errors is diminished

DNA extraction, library construction, hybrid capture, sequencing and sequence analysis

Can detect diverse activating High cost HER2 short variant mutations; can detect genomic alterations in the pathway are common in breast cancer, and may be predictive biomarkers for therapies targeting this pathway

[37,38]

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or FISH may be obtained, which are partially due to the variation in procedures made by different labs and partially stems from the inherent heterogeneity in distribution of HER2-positive tumor cells within the tumor [40]. Regarding these issues, several novel methods have been developed for HER2 testing in recent years. For example, mass spectrometry has emerged as a tool for HER2 testing because of its powerful quantification capability [41]. This chapter provides an overview of HER2 detection techniques with emphasis on those new methods. Notably, each method has its own advantages and disadvantages. There is still no consensus on which method is superior for assessing the HER2 status in breast cancer samples. These methods are discussed separately below.

4. Commercial assays 4.1 IHC Immunohistochemistry is an antibody-based, semiquantitative method used to evaluate HER2 protein expression typically on formalin-fixed paraffinembedded tissue samples. It is the most important primary technique for the determination of HER2 status. Briefly, slides are incubated with an antibody (e.g., A085 polyclonal antibody in the DAKO HercepTest assay_or the CB11 monoclonal antibody in the Ventana Pathway assay) directed against HER2. Then, excess antibody is washed off, and HER2 is finally made visible with a chromogen (e.g., diaminobenzidine, DAB) resulting in membrane staining. The more HER2 protein present, the stronger the staining. The amount of staining is usually scored according to 2018 ACSO guidelines as 0 (no staining is observed or membrane staining that is incomplete and is faint/barely perceptible and within
10% of tumor cells), 1 + (incomplete membrane staining that is faint/barely perceptible and within >10% of tumor cells), 2 + (weak to moderate complete membrane staining observed in >10% of tumor cells) or 3 + (circumferential membrane staining that is complete, intense, and within >10% of tumor cells) [20]. Breast tumors with absent or weak membrane staining (scored as 0 or 1 +) typically demonstrate a normal HER2 gene status and are regarded as negative, whereas cases scored as 2 + show poor agreement with the gene result and are considered equivocal [42,43]. All 2 + equivocal cases have to undergo a reflex test (same specimen using ISH) or a new test is ordered (new specimen if available, using IHC or ISH). An IHC 3 + score is interpreted as definite HER2 overexpression or HER2-positive; thereby,

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patients with a positive HER2 status at the IHC are eligible for targeted antiHER2 therapy, such as Herceptin, as described earlier [43,44]. Overall, IHC is easy to perform, relatively cheap and less time consuming. No specialized equipments are required [44]. More importantly, IHC detects the level of the HER2 protein, which represents the direct target of trastuzumab and other anti-HER2 therapies. Because staining results can be detected by a conventional bright-field microscope, the protein level can be evaluated in the context of tissue morphology [21]. Furthermore, slides can be stored because IHC provides permanent immunostaining. Although IHC testing is readily accommodated in most pathology laboratories, several factors may affect the quality of this assay, not only by choice of antibody but also by other determinants, such as tissue fixation, antigen retrieval, fixation time, embedding or slide storage used therein, and the choice of thresholds for reporting positive results [45,46]. Since interpretation is based on semiquantitative scoring, immunohistochemical analysis is susceptible to considerable interobserver variability and, therefore, to substantial discrepancies in IHC result interpretation, especially for the cases that are scored as 2 + [47]. Obviously, standardization of handling, fixation, quality control measures, and assessment of completeness are prerequisite to this IHC method and may help ensure accuracy and consistency for HER2 evaluation [48].

4.2 FISH The FISH test is not as widely available as IHC. However, FISH is generally considered more accurate [39,49]. In many cases, IHC is performed first, followed by FISH only if the IHC result is equivocal [50]. The widely accepted reference standard for determining HER2 status is FISH. Like IHC, FISH for HER2 testing is also morphologically driven, whereas it uses a fluorescently labeled probe to calculate the HER2 gene copy number within the nuclei of tumor cells on formalin-fixed, paraffin-embedded tumor samples. FISH can be conducted using either a single-probe or a dual-probe assay (using differentially labeled HER2 and chromosome enumeration 17 (CEP17) probes simultaneously) [51]. CEP17 is the centromere region of chromosome 17, which is the same chromosome on which HER2 is located, and, therefore, serves as a control. In most assays, the HER2 probe is labeled with an orange fluorophore, and CEP17 is counterstained with the green fluorophore, 4,60 -diamino-2-phenylindole (DAPI), or propidium iodide (PI). The probes are detected upon excitation of the fluorochromes and visualized using a fluorescence microscope. Single-probe assays give a

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direct copy number of the HER2 gene, while dual-probe assays calculate the relative copy number of the HER2 gene to that of CEP17 [24,25]. According to the ASCO/CAP guidelines published in 2018 [20] (Table 3), for the single-probe FISH assay, an average HER2 copy number <4.0 signals/cell indicates that HER2 is negative, an average HER2 copy number 4.0 and <6.0 signals/cell is considered equivocal, and an average HER2 copy number 6.0. For the dual-probe FISH assay, HER2 positive is defined as having a HER2:CEP17 ratio of >2.0 with an average HER2 copy number 4.0 signals/cell. Negative HER2 FISH amplification is defined as a HER2/CEP17 ratio <2.0 with an average HER2 copy number <4.0 signals/cell. The equivocal range for HER2 FISH assays is defined as three group: (1) a HER2/CEP17 ratio 2.0 with an average HER2 copy number <4.0; (2) a HER2/CEP17 ratio <2.0 with an average HER2 copy number 6.0 signals/cell; (3) a HER2/CEP17 ratio <2.0 with an average HER2 copy number 4.0 and <6.0 signals/cell. For equivocal results, a reflex test (same specimen using dual-probe ISH or using IHC) must be ordered and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH. Thus, FISH was routinely performed for clinical decision-making in the IHC 2 + group. According to the study of Slamon et al. with 900 cases of breast cancer patients, FISH may provide superior prognostic information for differentiating high-risk from lower-risk breast cancer [52]. In addition to accuracy, FISH is often considered as a more reliable, sensitive and reproducible technique for HER2 testing [25,53,54]. Since DNA is more stable than proteins, prolonged storage of paraffin blocks does not appear to affect its sensitivity, and preanalytical factors, such as tissue handling and fixation, have less impact on the assay results compared with IHC [55]. Furthermore, quantitative interpretation of FISH results with experience is relatively straightforward, and the concordance rate among observers is higher than with that of IHC. Another advantage of FISH testing is represented by the presence of internal controls. Inclusion of the CEP17 probe enables the recognition of CEP17 polysomy and therefore allows the distinction between pseudoamplification due to polysomy from true HER2 gene amplification [56]. However, this technique still has several shortcomings. FISH is more time-consuming and more expensive compared with IHC [43]. In addition, FISH testing requires costly equipment for signal detection and recognition, making it difficult to integrate the FISH assay in every routine diagnostic laboratory [57]. Since the morphologic aspects of tissue samples are difficult to evaluate under fluorescence, distinguishing invasive breast cancer from

Table 3 Summary of 2007, 2013 and 2018 guidelines and recommendations for optimal algorithm for HER2 testing. 2007 Recommendations 2013 Recommendations 2018 Recommendations

IHC

Positive: complete strongpositive membrane staining in >30% of tumor cells

Positive: circumferential membrane staining Positive: no change that is complete, intense, and within >10% of tumor cells

Negative: no staining or weak, Negative: no staining is observed or incomplete Negative: no change incomplete membrane staining membrane staining that is faint/barely in any proportion of tumor cells perceptible Equivocal: complete nonuniform membrane staining or with a weak intensity in >10% of the tumor cells FISH Positive: FISH ratio > 2.2 (dual probe)

Equivocal: circumferential membrane staining Equivocal: weak to moderate complete membrane that is incomplete and/or weak/moderate and staining observed in >10% of tumor cells within >10% of tumor cells or complete and circumferential membrane staining that is intense and within
10% of tumor cells Positive: FISH ratio 2.0 or ratio <2.0 with Positive: FISH ratio 2.0 with average HER2 copy average HER2 copy number 6.0 signals/cell number 4.0 signals/cell

Negative: FISH ratio <1.8

Negative: FISH ratio <2.0 with average HER2 Negative: FISH ratio <2.0 with average HER2 copy copy number 6.0 signals/cell number <4.0 signals/cell

Equivocal: FISH ratio 1.8–2.2

Equivocal: FISH ratio <2.0 with average HER2 copy number <4.0 signals/cell

Equivocal and need additional workup: (1) FISH ratio of 2.0 with average HER2 copy number <4.0 signals/cell; (2) FISH ratio of <2.0 with average HER2 copy number >6.0 signals/cell; (3) FISH ratio of <2.0 with an average HER2 copy number <6.0 signals/cell and 4.0

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breast carcinoma in situ is rather complicated. Furthermore, interpretation of FISH assays needs well-trained personnel [58,59]. Therefore, other ISH methods have emerged in recent years.

4.3 Bright-field ISH The power of ISH can be greatly extended by circumventing the fluorescence detection. Thus, bright-field ISH methods, such as chromogenic ISH (CISH), silver-enhanced ISH (SISH, i.e., mono-color SISH assay), and dual-color ISH (i.e., dual ISH) have been developed as alternative assays for FISH. For bright-field ISH, the sample preparation and probe hybridization steps are similar to FISH, and only the detection method differs. CISH makes use of a peroxidase enzyme-labeled probe for chromogenic detection using DAB [60], whereas SISH employs the same system with a silver-based detection system [61] and dual ISH combines the detection of HER2 gene copies via SISH assay, and CEP17 copies via CISH [62]. Since visualization is accomplished using a standard bright-field microscope, simultaneous analysis of gene copy numbers and morphologic features of tissue can be performed. Thus, the risk of nonmalignant compartment analysis can be reduced, and tumor heterogeneity can be recognized. For CISH testing, chromogenic signals are permanent, and thus, slides can be stored and used for retrospective studies. Compared with FISH, CISH is less expensive and less time-consuming, and it is easier to identify invasive components [63]. However, technical problems, including over- or underfixation of tissue samples, over or under digestion of tissue sections, and high background debris, can lead to erroneous results or loss of signal [64]. SISH is a fully automated bright-field ISH assay. The probes are visualized through the process of enzyme metallography [63]. The silver precipitation is deposited in the nucleus. Inter-observer reproducibility is more stable using the HER2/CHR17 ratio than HER2 copy number [65]. However, hybridization of another section is required for separate evaluation of CEP17 in monocolor SISH assay (i.e., single-probe SISH assay) [66]. Moreover, the SISH assay requires a rather expensive automated slide stainer that may not be available in many routine pathology laboratories [67]. Dual ISH assay allows the simultaneous visualization of both HER2 and CEP17 targets on the same slide [62]. This is very important, especially for the cases displaying CEP17 aneusomy or intratumoral heterogeneity. Although IHC and ISH have been FDA-approved for determining HER2 status in breast cancer, they have several disadvantages, especially false

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positives and false negatives in cancer management. As mentioned earlier, FISH was used as the gold standard for determining HER2 status. Thus, false-positive was defined as FISH negative but IHC positive and falsenegative was defined as FISH positive but IHC negative [20]. There is evidence indicating that amplification of the HER2 gene is the major mechanism leading to protein overexpression, accounting for approximately 97% of such cases [15]. Only in a very small proportion of cases, protein overproduction may result from transcriptional up-regulation. According to the results of the Kaufman PA study, false-negative rate is 4.0%, and their false-positive rate is as high as 20% [68]. False-negative results would deprive the patient of an important therapeutic option, whereas false-positive results would lead to an expensive ineffective treatment associated with unnecessary cardiotoxic side effects. Because of these, more and more novel methods for HER2 protein and gene detection have become available. Some of them will be introduced in this article.

5. Methods in development and not yet approved by FDA 5.1 New methods for HER2 protein expression 5.1.1 QD (quantum dot)-based probes Research on fluorescent semiconductor nanocrystals, known as quantum dots, has evolved over the past three decades from electronic material science to biological applications [69]. Several characteristics distinguish QDs from the commonly used fluorophores. QDs are single crystals of usually a few nanometers in diameter. Their size and shape can be precisely controlled by duration, temperature, and ligand molecules during the synthesis process [70]. This process yields QDs that have composition- and size-dependent absorption and emission. QDs usually have a broadband absorption spectrum in contrast to standard fluorophores [71]. In addition, the long fluorescence lifetime of QDs enables the use of time-gated detection to separate their signal from that of shorter-lived ones, such as background autofluorescence encountered in cells. Moreover, QDs tend to be brighter than conventional dyes because of the compounded effect of the extinction coefficients that are an order of magnitude larger than that of most dyes, whereas they have a comparable quantum yield and similar emission saturation levels [72]. Most importantly, their resistance to bleaching over long periods of time allows the acquisition of images that are well contrasted [69]. Another interesting property of QDs is the small number of QDs

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necessary to produce a signal. This means that single QDs can still be observed under immunocytological conditions with an ultimate sensitivity limit of one QD per target molecule [70]. The advances of QD-based probes have demonstrated a promising preclinical application for HER2 detection in recent studies [73,74]. In 2009, Chen et al. used a QDs-conjugated streptavidin (QDs-SA) probe for assessment of HER2 status in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue samples [75]. In this study, HER2 was first recognized by the monoclonal anti-HER2 antibody, followed by biotinylated anti-mouse IgG. Afterward, the probe bound to the IgG via a streptavidin-biotin interaction. The signal was ultimately detected at an emission wavelength at 605 nm. An IHC detection system was employed for analysis. Compared with conventional IHC, this QDs-SA probe approach is more accurate and economic, especially for cases of IHC 2 +. Its variation is lower than that of conventional IHC, which is attributed to the use of streptavidin-biotin labeling instead of diaminobenzidine (DAB) (Fig. 2). One year later in 2010, Chen et al. further developed an algorithm and a new parameter-total HER2 load for assessing HER2 status [76]. The total HER2 load was calculated by the following equation: Total HER2 load ¼ AFI  PPT  Tumor volume (AFI: the average fluorescence intensity; PPT: percentage of positive tumor cells). PPT is the ratio of distribution areas of HER2 to cytokeratin, and the tumor volume is defined as the cubic of the largest dimension of the tumor. The results indicated that this parameter may better reveal breast cancer heterogeneity and new subtypes of breast cancer

Fig. 2 Breast tumors with different HER2 IHC scores detected by (A) QDs-SA probe and IHC and (B) conventional IHC. Reprinted with permission C. Chen, J. Peng, H.S. Xia, G.F. Yang, Q.S. Wu, L.D. Chen, L.B. Zeng, Z.L. Zhang, D.W. Pang, Y. Li, Quantum dots-based immunofluorescence technology for the quantitative determination of HER2 expression in breast cancer, Biomaterials 30 (2009) 2912–2918.

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according to 5-year disease-free survival. This information could be helpful in formulating a more personalized targeted therapy for breast cancer. In 2014, Rakovich et al. used an oriented single domain anti-HER2 antibodies (sdAbs)-QD conjugate for HER2 detection with confocal microscopy imaging [77] (Fig. 3). Compared with the performance of antibodies conjugated to conventional organic dyes, such as Alexa Fluor 488 and Alexa Fluor 568, the sdAbs-QD conjugate was engineered with all the sdAb antigen recognizing sites facing outward. This approach allowed selective staining in a panel of breast cancer cell lines with differential HER2 expression. Thus, these nanoprobes hold a great potential for further development of clinical HER2 assays. Recently, in 2018, P
ereztrevin˜o et al. explored a multiplexed imaging method using affibody-quantum dot (Aff-QD) conjugates, ratiometric analysis (RMAFI) and breast cancer multicellular tumor spheroids (BC-MTS) as a 3D model of breast cancer [78]. QD-based probes have been developed upon conjugation with novel synthetic, molecular recognition proteins, called affibody molecules. Confocal XY sections were recorded along the Z distance and processed by automatic RMAFI. HER2 can be detected up to 30 μm within intact BC-MTS, and permeabilization can further improve the detection up to 70 μm (Fig. 4). 5.1.2 Mass spectrometry (MS) As described above, most of the developed assays provide only a limited degree of qualitative data for HER2 protein expression. Fluorophore and chromosome studies rely heavily on the subjective judgment of researchers, and samples with slight differences are not easy to distinguish. Therefore, quantitative assays are greatly urged. During the past 20–30 years, mass spectrometry has migrated from the research realm into the clinical laboratory [79]. MS is now routinely used in many clinical laboratories for quantification of a huge range of low molecular weight compounds (normally <1500 Da) in biological samples [80,81]. Recently, technological advances in mass spectrometry have given rise to instruments and methods that are fully capable of automated and highthroughput protein assaying [82]. Traditionally, there are two primary MS approaches for proteins: bottomup and top-down [83]. As one of the recognized bottom-up methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics can quantify target proteins with a high sensitivity, a high selectivity, and a wide dynamic range [84]. Specific detection and determination of

Fig. 3 Structural representation of antibodies and sdAbs-QD conjugates. Reprinted with permission T.Y. Rakovich, O.K. Mahfoud, B.M. Mohamed, P.M. Adriele, C.S. Kieran, V.D.B. Tina, D.K. Line, S. Alyona, B. Daniel, R. Aliaksandra, Highly sensitive single domain antibody-quantum dot conjugates for detection of HER2 biomarker in lung and breast cancer cells, ACS Nano 8 (2014) 5682–5695.

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Fig. 4 Representative optical sections acquired at a depth of 50 μm within the nonpermeabilized and permeabilized BC-MTS (left panels), and their respective orthogonal view (right panels). Reprinted with permission P.T. Perla, D.L.C. Hernández, J. P
erez-Treviño, O.R. Fajardo-Ramírez, N. García, J. Altamirano, 3D imaging detection of HER2 based in the use of novel affibody-quantum dots probes and ratiometric analysis, Transl. Oncol. 11 (2018) 672–685.

a protein of interest at the peptide level are the key concepts of this targeted analysis. Surrogate peptides are produced via enzymatic digestion of the target protein and monitored using selected/multiple reaction monitoring (SRM/ MRM) [84]. In contrast, the “top-down” proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating the integral protein in the mass spectrometer. It requires more complex instrumentation and methodology than the “bottom-up” approach. Among the available techniques, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. This method can simultaneously determine the distribution of hundreds of analytes in a single measurement without any need for labeling. To date, MALDI-IMS

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has been applied for the analysis of a number of proteins and multiple types of diseased tissues [85,86]. In the case of HER2, mass spectrometry-based approaches may encounter unique challenges for membrane proteins, predominantly derived from their hydrophobic and proteolytically resistant nature [87]. Insufficient solubility and low digestion efficiency could be confounding factors. Therefore, mass spectrometry has been rarely used for the detection of HER2 status, and relevant research is very scarce up to now. However, with the further development of IMS and the novel application of targeted proteomics, unique information for HER2 could be obtained. In 2009, Rauser et al. demonstrated that use of MALDI-IMS can provide a classification model for HER2 status in breast cancer tissue [14,85]. Sinapinic acid at 10 mg/mL in water/acetonitrile at 40:60 (v/v) with 0.2% trifluoroacetic acid was used as the matrix, and the HER2 protein profile was obtained through direct analysis of tissue (Fig. 5). The proteomic signature was able to accurately define HER2-positive from HER2-negative tissue samples, achieving high values for sensitivity at 83%, for specificity at 92% and an overall accuracy of 89%. In 2015, Steiner et al. developed an SRM assay for HER2 [86]. The six best surrogate peptides were quantified and evaluated for linearity, precision and lower limit of quantification (LLOQ). Breast cancer tissue samples were digested through heating, sonication and overnight tryptic digestion. The assay showed good analytical performance and a high agreement with IHC and FISH data. While direct digestion of HER2 seems complex and time-costing, a quasi-targeted proteomics assay has been developed. In 2018, Chen et al. have developed a quasi-targeted proteomics assay and converted the HER2 signal into the mass response of reporter peptide by a combination of aptamer-peptide probe and LC-MS/MS [41] (Fig. 6). Notably, the aptamer-based HER2 detection techniques developed so far are mostly built on a fluorescent or electrochemical sensing platform. In that study, the aptamer-peptide probe was composed of the anti-HER2 aptamer HB5 and the selected substrate peptide GDKAVLGVDPFR. The Kd of the probe was estimated to be 29.5 nmol/L. GDKAVLGVDPFR was composed of the reporter peptide AVLGVDPFR and 3 amino acid peptides (GDK) containing a lysine residue at the carboxyl terminus linked with the reporter peptide. The probe recognized and bound to HER2 followed by trypsin surface shaving. The reporter peptide was finally released and quantified

Fig. 5 Images of (A) HER2-positive and (B) HER2-negative breast tumors. Reprinted with permission S. Rauser, C. Marquardt, B. Balluff, S.O. Deininger, C. Albers, E. Belau, R. Hartmer, D. Suckau, K. Specht, M.P. Ebert, Classification of HER2 receptor status in breast cancer tissues by MALDI imaging mass spectrometry, J. Proteome Res. 9 (2010) 1854–1863.

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Fig. 6 Schematic representation of aptamer–peptide probe preparation and its combination with quasi-targeted proteomics for HER2 detection.

using an LC-MS/MS-based targeted proteomics assay. In this way, the HER2 signal can be converted into the level of reporter peptide. The resulting limit of quantification for HER2 was 25 pmol/L. Moreover, HB5 showed relatively strong binding to HER2-positive cells (BT474 and SK-BR-3), whereas HB5 binding to HER2-negative cells (MDAMB-231 and MCF-7) was weak. Most importantly, the result of tissue test was highly concordant with that of IHC with reflex testing FISH (κ ¼ 0.880).

5.2 New methods for HER2 gene detection 5.2.1 Next generation sequencing (NGS) for HER2 detection NGS, also known as massively parallel sequencing, was developed based on the traditional Sanger-type sequencing method [88,89]. In NGS, the genomic strand is fragmented, and the bases in each fragment are identified by emission signals when the fragments are ligated against a template strand. The advantage of NGS is that it uses array-based sequencing to process millions of reactions in parallel, resulting in very high speed and throughput at a reduced cost. It has three general steps: library preparation, amplification and sequencing. There are some commercial NGS platforms, including Illumina, 454 Pyrosequencing, SOLiD, Ion Torrent and others [90].

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For gene detection, NGS has a more important feature, providing millions of much shorter reads (21 to 400 base pairs). These short reads from each DNA molecule can be counted and quantified in the sequencing. Therefore, copy number assessment of each genomic region is accurate [91]. In the case of HER2 gene detection, FoundationOne CDx™ (F1CDx), an NGS device, has been approved by the FDA. The experimental procedure principally includes DNA extraction, library construction, hybrid capture, sequencing and sequence analysis. During sequence analysis, HER2 gene amplification is detected using a comparative genomic hybridization (CGH)-like method. First, a log-ratio profile of the sample is acquired by normalizing the sequence coverage obtained at all exons and genome-wide SNPs (3500) against a process-matched normal control. This profile is segmented and interpreted using allele frequencies of sequenced SNPs to estimate the tumor purity and copy number at each segment [38]. Lipson et al. tested 35 FFPE invasive breast carcinomas and found that the result had 97% accuracy relative to that determined by FISH [92]. In addition, NGS can also identify HER2 short variant mutations [93]. However, if tumor purity is <25% in the samples, sensitivity for the detection of copy number alterations (CNAs) in HER2 may decrease [94]. 5.2.2 Polymerase chain reaction (PCR)-based assays for HER2 detection PCR is a primer-mediated enzymatic amplification of specifically cloned or genomic DNA sequences. Currently, this PCR process is automated for routine use in laboratories worldwide [95,96]. PCR normally has three simple steps: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers [97]. This method uses endpoint measurement in which the target gene is measured after a fixed number of cycles; then, the PCR product is analyzed using fluorescence detection. Multiplex ligation-dependent probe amplification (MLPA) is a recently developed PCR method used to detect copy number variation. MLPA evaluates gene amplification by amplifying added oligonucleotide probes instead of sample nucleic acids [98]. The probe contains two fractions. One is composed of a target-specific sequence and a universal primer sequence, and the other contains a target-specific sequence, a stuffer sequence of different lengths and sequence and a universal primer sequence. Once the two fractions recognize target sequences, a ligase works to link the two factions

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Fig. 7 Outline of the MLPA reaction. Reprinted with permission J.P. Schouten, C.J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, and G. Pals, Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification, Nucleic Acids Res. 30 (2002) e57.

together as a whole. As a result, the universal primer sequences of the probe can be amplified by PCR, whereas unbound probe halves cannot be amplified. Due to a unique length of a complete probe, the PCR products can be separated and identified by capillary electrophoresis [98,99] (Fig. 7). Moerland et al. used MLPA for HER2 detection, and the result showed that the overall agreement between FISH and MLPA was 98% [100].

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The MLPA assay is fast, accurate and inexpensive. When performing MLPA, only small quantities of DNA are required, and fragmentation of DNA does not have an impact on the reliability of the results [89]. In addition, it can overcome the inherent problem of IHC and FISH, such as subjectivity and failure to provide quantitative data. However, copy number changes detected by MLPA should always be confirmed by other methods because some probe signals may be affected by factors, such as sample purity and small changes in experimental conditions [98]. Furthermore, tissue morphology is not conserved using this assay, and tumor heterogeneity can be missed [101]. In addition to these, design and preparation of an MLPA probe mix are difficult and time consuming [98,102]. Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression. Specific interests have been exerted using methods for detecting and determining the abundance of a particular mRNA in a total or poly (A) RNA sample. mRNA ISH is similar to CISH as described above, but the probes are different. In CISH, the DNA probe hybridizes to DNA, whereas in mRNA ISH, the probe hybridizes to the mRNA of HER2. Currently, there is an available kit called HistoSonda (Cenbimo, Lugo, Spain) to detect mRNA transcribed from the HER2 gene in FFPE breast cancer samples. The probe consists of 2 fragments of single-stranded DNA with lengths of 694 and 1143 nucleotides that are targeted against the HER2 gene. After protein digestion by proteinase K, the digoxigenin-labeled single-stranded DNA probes are hybridized with HER2 mRNA. Subsequently, an anti-digoxigenin antibody and an HRP labeled secondary antibody are joined. A cell cytoplasmic stain is considered positive, whereas no stain is interpreted as a negative result. In the study of Laia, the overall agreement between CISH and HistoSonda was 89% for HER2 detection in FFPF tissue [103,104]. Moreover, David et al. called this method rapid in situ hybridization (RISH) [105], and the comparison result showed that agreement between HER2 CISH and RISH™ methods was 98%. Compared with IHC and FISH, this assay is simple, fast, specific and cost-effective. However, its shortcomings are similar to other ISH assays: it is semiquantitative. In addition, it has the issue of mRNA degradation [103,106]. The characteristics of the assays for HER2 gene detection have been summarized in Table 4.

Table 4 Main characteristics of the HER2 gene detection assays. Method of Specimen analysis type Detection mechanism Advantages

Limitations

NGS

FFPE tissue

Fragments of the genomic strand are amplified. After that, the fragments are sequenced and quantified using a comparative genomic hybridization (CGH)-like method

Samples with <25% tumor may High speed and throughput, allowing the have decreased sensitivity for the accurate copy number assessment of each detection of CNAs genomic region, identifying ERBB2 short variant mutations and genomic alterations in the HER2 downstream pathway

MLPA

FFPE tissue

Target sequence in the probe hybridizes with target DNA, then the probe is amplified by PCR reaction. The PCR products can be separated and identified by capillary electrophoresis

Fast, accurate, and inexpensive, only small quantities of DNA extracted from paraffinembedded materials are required, fragmentation of DNA does not have an impact on the reliability of results, yield quantitative results, overcome subjectivity of IHC and FISH, quantify HER2 amplification

mRNA FFPE ISH tissue

A HRP-labeled probe hybridization Simple, faster, higher specific, cost-effective and can eliminate the confusing aspects of with target tumor mRNA, and IHC made visible with diaminobenzidine, and detected by bright-field microscope

Should always be confirmed by other methods, tissue morphology is not conserved and tumor heterogeneity can be missed, design and preparation of an MLPA probe mix are difficult and time consuming Semi-quantitative, mRNA is easy to degrade

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6. Detection of the HER2 protein in serum samples Both the IHC and ISH methods lack “real-time” follow-up [107]. In clinics, these methods are mainly applied to tumor biopsies. As patients who were diagnosed with HER2-positive breast cancer and are undertaking treatment return for follow-up testing, the HER2 status is not routinely retested. However, the clinician may need to know whether the HER2 status of the tumor has changed and subsequently alter the treatment at times. Within this context, neither IHC nor ISH is a practical assay [31]. In contrast, patient serum may be a good option for real-time testing. Currently, it is well known that the extracellular domain (ECD) of the HER2 protein is cleaved and released into the circulation. Serum concentrations of HER2 are elevated in 20%–50% of patients with primary breast cancer and in 50%–62% of those with metastatic disease [108]. Normal individuals usually have a HER2 concentration between 2 and 15 ng/mL in serum, whereas breast cancer patients have serum HER2 levels in the range of 15–75 ng/mL [109]. Moreover, serum collection is noninvasive, which may reduce patient suffering from biopsy. To date, several studies have suggested that serum HER2 could be used as a biomarker for monitoring the disease course and the patient’s response to therapy [110,111]. However, the clinical usefulness of serum HER2 has not been fully validated because of conflicting data [31]. The enzyme-linked immunosorbent assay (ELISA) test was the first serum HER2 testing assay that was approved by the FDA in 2000 [112]. Since then, a few methods have been developed for serum HER2 testing.

6.1 ELISA ELISA is a commonly used analytical biochemistry assay in the clinic and lab. The assay uses a solid-phase enzyme immunoassay to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. Generally, serum HER2 is measured by using two monoclonal antibodies recognizing two distinct epitopes of the antigen. First, a monoclonal antibody target to HER2 is coated on 96-well plate. After binding to HER2, the immobilized protein is then incubated with the second monoclonal antibody, which is labeled with HRP in advance. After application of the HRP substrate, detection is accomplished by assessing the colored end product with spectrophotometry, which

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correlates to the HER2 concentration in the sample [113]. There are some commercially available ELISA platforms for HER2 ECD detection, including an automated platform (Immuno-1), a microtiter plate format and ADVIA Centaur from Siemens Healthcare Diagnostics [31]. ELISA is a quick, simple and less invasive assay for quantification. In addition, ELISA can be used to monitor the dynamic changes of HER2 status over the course of the disease progression or following treatment. However, the results obtained by ELISA may not be reliable if the serum samples are from patients receiving trastuzumab treatment since trastuzumab that is still present in the patient’s serum may compete with the two antibodies used in the assay [31,99,114].

6.2 Surface plasmon resonance (SPR) Since the first demonstration of SPR in the early 1980s, SPR sensors have made vast advances in technology and its applications [115]. SPR is the resonant oscillation of conduction electrons at the interface between a negative and positive permittivity material stimulated by incident light. SPR is the basis of many standard tools for measuring adsorption of a material onto planar metal (typically gold or silver) surfaces or onto the surface of metal nanoparticles [116]. The most significant advantages of SPR include label-free sensing and enabling the direct detection of target analytes, in addition to portability, making it suitable for point-of-care (POC) analysis. To date, SPR sensing has been applied to detect a series of biomolecules. In 2017, Monteiro et al. proposed an SPR biosensor to assess serum HER2 using nanohole arrays on a gold thin film by signal transduction of transmitted light measurements from array image acquisitions [117]. These metallic nanostructures may directly couple with the light on the surface plasmon using a simple collinear arrangement. The proposed device reached an average sensitivity of 3 ng/mL HER2 (Fig. 8).

6.3 Electrochemical detection Electrochemical detection studies the relationship between electricity as a measurable and quantitative phenomenon and the identifiable chemical change with either electricity considered as the outcome of a particular chemical change or vice versa. These reactions involve electric charges moving between electrodes and an electrolyte (or ionic species in a solution). Thus, electrochemical detection deals with the interaction between

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Fig. 8 Schematic representation of the HER2 detection with SPR sensing. Reprinted with permission A. Aub
e, S. Campbell, A.R. Schmitzer, A. Claing J.F. Masson, Ultra-low fouling methylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates, Analyst 142 (2017) 2343–2353.

electrical energy and chemical changes [118]. Electrochemical sensors have provided fast, sensitive and specific detection of serum HER2 with a low detection limit. In 2008, Capobianco et al. demonstrated the real-time, label-free detection nature of HER2 detection using a MPS-coated glass piezoelectric microcantilever sensor (PEMS) with the single-chain variable fragment, scFv, immobilized on the sensor surface [119]. A concentration of 5 ng/mL of serum HER2 can be determined in a background of 1 mg/mL of BSA. In 2013, Chun et al. developed a novel impedance aptasensor by immobilizing a HER2-specific single stranded DNA (ssDNA) aptamer onto a monolayer of 3-mercaptopropionic acid self-assembled on a gold nanoparticle electrode [120]. The response of the aptasensor showed a proportional relationship with the concentrations of serum HER2 ranging from 105 to 102 ng/mL. The aptasensor also showed fast HER2 detection with negligible cross-reactivity to various compounds that are likely to exist in human serum samples (e.g., glucose, IgG, DNA and RNA). Moreover, the aptasensor can be regenerated by a simple pH-shift method. Two years later in 2015, Qureshi et al. developed a label-free capacitive aptasensor based on capturing the HER2 protein by anti-HER2 ssDNA aptamers functionalized on interdigitated microelectrodes of a capacitor as biorecognition elements [121]. The aptasensor response was measured by nonfaradaic impedance spectroscopy (nFIS) method. As a result, the HER2 protein was successfully detected through concentration dependent changes in impedance/capacitance values as a result of the formation of the

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aptamer-HER2 protein complex (0.2–2 ng/mL of HER2) on capacitor microelectrodes. Recently, Li et al. developed a polycytosine DNA-based immunosensor for electrochemical detection of HER2 [122]. They utilized gold nanoparticles (AuNPs) as a supporting matrix to immobilize polycytosine DNA sequence (dC20) for electrochemical current generation and antiHER2 antibodies. In the presence of the target HER2, a sandwiched immunocomplex forms between a peptide specific to HER2 that is immobilized on the gold electrode and the anti-HER2 antibodies on the AuNPs, generating an electrochemical current on the surface of the electrode. The calculated limit of detection of HER2 was as low as 0.5 pg/mL, and the detection was linear to HER2 from 1 pg/mL to 1 ng/mL. There is no cross-reactivity with several potential interferences, such as human IgG, human IgA, p53, carcinoembryonic antigen, and protein kinase. The sensor’s performance with HER2 in clinical serum samples is similar to that of commercial ELISA assays as mentioned earlier.

6.4 QD-based detection QDs combined with fluorescence resonance energy transfer (FRET) can also be used to test HER2 in serum. Hong et al. constructed a new and enhanced FRET biosensing platform by employing a novel donor-acceptor pair [123]. In the platform, MnCuInS/ZnS QDs encapsulated in BSA were ideal donors, and urchin-like AuNPs were selected as the energy acceptors. Then, the donors were modified with HER2 aptamers, and the acceptors were conjugated with another aptamer that was partially complementary with the HER2 aptamer. Then, FRET occurred by the binding between MnCuInS/ZnS@BSA-Apt and the urchin-like AuNPs-ssDNA. In the presence of HER2, the FRET effect was broken and fluorescence increased (Fig. 9). The result exhibited a wide detection range (2–100 ng/mL) and a low detection limit (1 ng/mL). Qiu et al. used FRET and QDs to develop a homogeneous and multiplexing immunoassay for HER2 together with other protein biomarker detection [124]. The detection limit was 8.0 ng/mL for HER2).

6.5 Mass spectrometry Currently, rare studies have been performed using mass spectrometry to detect serum HER2, possibly due to the complexness of serum samples. Compared with tissue samples, serum is the richest and the most complete

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Fig. 9 Schematic illustration of the enhanced FRET system toward HER2 detection on the basis of NIR MnCuInS/ZnS@BSA and urchin-like AuNPs. Reprinted with permission H. Xing, T. Wei, X. Lin, Z. Dai, Near-infrared Mncuins/Zns@BSA and urchin-like Au nanoparticle as a novel donor-acceptor pair for enhanced FRET biosensing, Anal. Chim. Acta. 1042 (2018) 71–78.

informative proteome. It contains 60–80 mg of protein/mL. Of these proteins, albumin comprises 50%–55% of the serum protein content. Along with albumin, only a few other proteins (e.g., immunoglobulin) constitute 90% of the total protein content of serum. Almost all the remaining 10% is made up of 12 proteins. These abundant proteins can significantly affect the resolution and sensitivity of many techniques such as MS, leading to difficulties in the detection of low-abundant proteins that are of clinical importance. Even though serum can be made significantly less complex by depleting proteins in high abundance, HER2 levels (<75 ng/mL) still easily fall below the assay detection limit. In this situation, efficient sample pretreatment approaches including isolation and enrichment such as molecularly imprinted polymers (MIPs) and protein/peptide immunoaffinity enrichment could be employed [125,126].

7. Conclusions Accurate assessment of the HER2 status of a tumor is critical for early diagnosis and successful treatment. While IHC for protein expression and ISH for gene amplification have been approved by the FDA and strict following of these established guidelines and procedures could greatly avoid

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misclassification of patients and improve the clinical outcome, novel quantitative methods are needed to significantly enhance clinical decision support. Finally, the assays reviewed here are actually not limited to the specific biological specimens presented herein. Some of them can be easily adapted to others after slight alterations.

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