A review on methods for diagnosis of breast cancer cells and tissues

Abstract Breast cancer has seriously been threatening physical and mental health of women in the world, and its morbidity and mortality also show clearly upward trend in China over time. Through inquiry, we find that survival rate of patients with early‐stage breast cancer is significantly higher than those with middle‐ and late‐stage breast cancer, hence, it is essential to conduct research to quickly diagnose breast cancer. Until now, many methods for diagnosing breast cancer have been developed, mainly based on imaging and molecular biotechnology examination. These methods have great contributions in screening and confirmation of breast cancer. In this review article, we introduce and elaborate the advances of these methods, and then conclude some gold standard diagnostic methods for certain breast cancer patients. We lastly discuss how to choose the most suitable diagnostic methods for breast cancer patients. In general, this article not only summarizes application and development of these diagnostic methods, but also provides the guidance for researchers who work on diagnosis of breast cancer.

lifestyle environments and so on. 4,6,7 The high incidence rate of BC is unavoidable, but decreasing the mortality of BC is feasible. Early detection and treatment are critical to curing BC, because it tends to metastasize in the middle and last stage. [8][9][10] Therefore, finding BC is vital in early stage, which can greatly improve the survival rate of patients.
To quickly and accurately screen BC, many diagnostic methods based on imaging and molecular biotechnology have been developed. It is indispensable to summarize and evaluate these methods, to provide value information for clinical diagnosis. Jafari 11 summarized various imaging techniques and biochemical biomarkers used for detection and monitoring BC patients and highlighted that it is helpful to diagnose and treat patients with BC by measuring level of certain biomarkers. Weaver 12 described definitions and applications of imaging "biomarkers," and thought that they can build the decision support system by these markers to provide help for clinical breast care and BC-related research. Many articles review these methods for diagnosing BC mainly from these aspects, by introducing the contribution of imaging techniques (including molecular imaging markers) in diagnosing BC patients, and summarizing these findings on connection between newly discovered tumour makers and BC patients. [13][14][15] Many articles describe a large number of diagnostic methods for breast cancer, but few articles introduce how to choose suitable diagnostic methods for different types of BC patients.
In this review, many diagnostic methods are reviewed, such as mammography (MG), ultrasonography (US), magnetic resonance imaging (MRI), nucleic acid hybridization system (NAHS), real-time fluorescence quantitative PCR system (RT-qPCR), protein hybridization system (PHS), flow cytometer (FCM) and so on. We herein introduce their development and summarize their advantages and disadvantages and provide some diagnostic schemes for different types of BC patients.
The article can help future research and development in diagnosing BC patients and guiding people who are working on BC research, on how to choose the suitable methods for diagnosing BC patients.

| IMAG ING D IAG NOS IS
Utilization of imaging techniques shows clearly the morphology and location of tumour tissues and proves much clinical information that is valuable to doctors. However, imaging techniques may cause harm to patients when using contrast agents and high energy rays. Therefore, we should discuss these imaging techniques and choose the most appropriate diagnostic method for BC patients. single-photon emission computed tomography (SPECT). In Table 1, we list the advantages and disadvantages of these imaging methods. In view of high cost, poor practicability and radiation damage, PET, CT and SPECT are not recommended in diagnosing BC patients.
However, these techniques can be used as auxiliary diagnostic methods for diagnosing BC in some special cases, such as screening for metastatic BC, presence of bone and lymphatic metastases.
Therefore, we only introduce MG, US and MRI that are preferred methods for screening BC. Summary and evaluation of these common imaging techniques will help doctors to better serve patients and promote the development of clinical diagnosis.

Imaging techniques
Advantages Disadvantages | 3 of 16 HE Et al.

| Mammography
Mammography (MG) is preferred strategy for screening and diagnosing BC and helps doctors obtain clinic information on BC patients.

| Magnetic resonance imaging
Magnetic resonance imaging (MRI) allows early detection of familial BC regardless of patients' age, breast density or risk status. 39 Figure 1 is schematic diagram of MRI.

| Novel specific biomarkers
Circulating tumour cells (CTCs) enter the blood circulation from primary tumour tissues, and the number of CTCs is about 1 ~ 10 2 / mL in peripheral blood. Jin et al 51 investigated the viability of using CTCs could be used as a novel biomarker in assisting BC detection. In all, these novel biomarkers not only are monitored dynamically, but are also used to judge prognosis. The patient's body fluids are used as samples for CA biopsy.

| Nucleic acid hybridization
Nucleic acid hybridization techniques mainly include fluorescence in situ hybridization (FISH) and aptamer probe hybridization (APH). is considered the "gold standard" for detecting whether the HER-2 gene is activated. 65 In addition, FISH shows other advantages, including reproducibility, stability and high sensitivity. However, these factors limit its promotion, including the need for complex probes design and special fluorescence detector. In the future, multicolour fluorescence in situ hybridization will be a tendency in greatly improving the throughput when searching genetic sites.
Aptamer probe hybridization is another highly sensitive and specific technique. Suitable aptamers are key factors in the accuracy of APH. These aptamers mainly are produced by Systematic Evolution of Ligands by Exponential enrichment (SELEX). 66 At present, Cell-SELEX is one of the most representative of SELEX, and it has become the main method that gets optimal aptamers from tumours. 67 The schematic diagram of Cell-SELEX is shown in Figure 3. Suitable aptamers can identify some specific fragments that can be used to diagnose diseases. Kim 68 prepared a nucleotide aptamer (SE15-8-QDs) for detecting BC and found that it is more sensitive than the and is not suitable for promoting to use in primary hospitals. In the future, APH will have the easy process for screening suitable aptamers and will find more biomarkers of BC.
F I G U R E 2 Technical principle of fluorescence in situ hybridization

| Gene chip and next-generation sequencing
Gene chip can analyse a large number of fragments of nucleic acid simultaneously, and it is applied widely in diagnosing BC. Gene chip is used to observe and analyse the condition of nucleic acids in BC cells or tissues and also can find new diagnostic biomarkers for BC by screening a large number of samples. As well known, gene chip is essentially a high-density oligonucleotide microarray. [70][71][72] At present, there are two methods for chip preparation: in situ synthesis and direct point method. 73 However, in situ synthesis is the main method, and its schematic diagram is shown in Figure 4. Using gene chip technology, researchers found mechanisms for doxorubicin resistance in BC and screened these key genes for BC therapy. 74  In the future, with development of nanotechnology, the size of chip will be smaller and the throughput of gene chip will be higher.
Next-generation sequencing (NGS) was put forward by Metzker. 76 The schematic diagram of NGS is shown in Figure 5.  Gene promoter region DNA methylation can also cause cancer, because it can produce similar effects to gene mutations, such as obtaining or losing functions of some specific genes. 93,94 Methylation-based RT-qPCR system is widely used for analysing genetic methylation.

| Real-time fluorescence quantitative PCR system
The Table 2 lists part of methylation genes in BC. In order to understand the detection process for methylated genes, we elaborate it by However, MethyLight has some limitations; for example, the nucleic acid needs to be treated (totally methylated or un-methylated nucleic acid), needs to design complex probes and requires professional operation. In the future, integrating extraction and methylation detection of DNA will be a tendency, which will not only improve the DNA yield, but also the efficiency of methylation.

| Protein hybridization system
Tumour cells or tissues contain not only the nucleic acids but also many proteins. The "central dogma" of molecular biology shows that proteins are closely associated with nucleic acids. However, if the final protein has no change, the differential expression of nucleic acids may not cause cancer. Therefore, proteins are another important biomarker for diagnosing cancers and analysing the situation of proteins can predict occurrence of cancer. Similarly, proteins, as important biomarker, make great contribution to diagnosis of BC. In Table 3, we introduce the most common and latest oncogene proteins involved in BC. These proteins can be quantitatively evaluated by immunochemistry, RT-qPCR and Western blot. The difference between them is the different detective object, in which RT-qRCR is for mRNA and Western blot and immunostaining are for proteins.

| Immunochemistry
For pathologists, immunostaining (IHC) can accurately locate the site of organization and is an auxiliary method for diagnosing BC.

PITX2
PITX2 gene is a prognostic marker for progesterone receptor-positive patients, and it is closely associated with poor survival and distant metastasis of breast tumours. If PITX 2 gene is methylated, it can be considered low risk of distant metastasis recurrences and need not adjuvant chemotherapy 150 Hox The methylation of Hox gene is closely related to the high expression of oestrogen and progesterone receptors, and methylation of HoxD13 gene is closely related to breast tumour size and poor clinical treatment 151

AKT1
Methylation of AKT1 gene is observed to be associated with BC, and it affects expression of AKT1 gene. The expression of AKT1 gene has significantly associated with HER-2 protein status 152

Soxl7
Soxl7 gene has significantly associated with breast tumour size and lymphatic metastasis, but un-methylation of Soxl7 gene is found in normal breast tissue and serum 153

CDKN2A
The methylation of CDKN2A gene in patients with malignant tumour is found, but un-methylation of CDKN2A gene is found in patients with benign breast disease. Methylation of CDKN2A gene also is associated with distant metastasis of breast tumours 154 FHIT FHIT gene is widely expressed in normal tissues, and methylation of FHIT gene occurs in 31% of patients with primary BC. In particular, after FHIT gene is methylated, its expression quantity is changed in patients with sporadic ductal carcinoma 155

TIMP-3
Methylation of TIMP-3 gene is found in BC cells, but does not find in normal tissues. The degree of methylation of TIMP-3 gene is positively correlated with malignancy of BC 156

MDGI
The MDGI gene is also lowly expression in BC tissues. If promoter region of MDGI gene is methylated in breast cancer patients, methylation of MDGI will be only slightly influenced by surgery, whereas tamoxifen therapy will be a more pronounced effect 157

RASSF1A
In the patients with sporadic BC, finding 33.3% of RASSF1A gene was deleted or methylated 158

HSD17B4
Methylation of HSD17B4 gene is an independent predictive marker for pathological complete response in some studies. If the HSD17B4 is not methylated in patients with BC, these patients will be not benefit from trastuzumab treatment, but will be benefit from lapatinib treatment 159

ESR1
Abnormal hyper-methylation of ESR1 gene is found in BC cells, and it will hope to become a new biomarker of breast tumour 160

RhoBTB2
Aberrant methylation of RhoBTB2 gene may affect expression of the RhoBTB2 gene, which influences PR protein status, become the factor that induce BC 161

NBPF1
Hypermethylation of promoter region of NBPF1 gene is found in patient's serum or plasma with BC, and thus, the NBPF1 methylated from patient's serum or plasma may become potential tumour biomarker for detection of BC 160 IHC analysis of breast tumours has advantages in the following four  formation may cause changes of protein levels, so IHC is able to study the mechanism of breast tumours by analysis of protein levels. However, IHC needs fluorescence labelling which is time-consuming and difficult to prepare.

| Western blot
Similarly, Western blotting also utilizes the antigen-antibody binding character that is highly specific. On one hand, the capacity of histological localization of Western blotting is poorer than that of IHC, but the capacity of quantitative protein level is more accurate than that of IHC. On the other hand, for RT-qPCR, though they all evaluate quantitatively the level of proteins, their detection objects are different, where RT-qPCR is for nucleic acids, and Western blotting is for proteins. Using exogenous proteins to study proteins' interaction is a common approach, but the most rigorous approach is to detect interactions between endogenous proteins. 107    the mechanism of inhibitory and cytotoxic activity of anticancer agent on BC cells, cell cycle progression using flow cytometry analysis, and found some valuable medicine.

| Flow cytometer
There are some advantages of FCM, including nonspecific binding in antigen antibody may cause the signalling pathway of FCM to be affected. Dyestuff pollution in FCM experiment is also a big trouble, and expensive instruments are required. In the future, it is most important that diagnostic scheme for FCM should be standardized and agents of high efficiency and low cost should be developed.

| Puncture biopsy system
Needle biopsy is a main method to obtain tumours tissue or cells sample for histopathological diagnosis. These puncture biopsies system include fine-needle aspiration cytology (FNAC), core needle biopsy (CNB) and vacuum-assisted breast biopsy (VABB). 129 However, there are some disadvantages of needle breast biopsy; for example, it may cause tumours transfer and researchers thought that high-grade, non-coaxial biopsies, triple-negative BCs and multiple insertions may be risk factors for neoplastic seeding. [134][135][136][137] In the future, with development of biopsy needle, the risk of neoplastic seeding will be reduced and the accuracy of diagnosis will be improved. Surely, the latest imaging guidance will also promote the development of puncture biopsy.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
In this review, we mainly introduced the common methods for diagnosis of BC. As the exploration of imaging technology goes deeper, researchers realize that the single imaging technology has F I G U R E 8 Isolation of individual droplets by flow cytomtry. 121 (Reproduced with permission from Copyright 2020, American Chemical Society) lower accuracy and cannot meet the need for BC diagnosis, and the combination of various imaging modalities will be one of the major developing directions. [138][139][140] Moreover, with development of biosensors, a lot of BC biomarkers have been found. The combination of imaging sensors and biosensors can get unexpected results. 141,142 Meanwhile, more and more aptamers are developed, which increases connection between imaging and molecular biology. 143,144 These aptamer-functionalized nano-composites not only can become indicators for imaging, but also can identify cancer cells, and/or even classify BC cells subsets. In another aspect, screening for new tumour biomarkers is still an important task which can help doctors diagnose BC faster and more accurately.
Currently, proteins, nucleic acids and lipids are the main tumour markers in breast cancer, while the question remains whether single markers could not acquire definite diagnosis results. 145,146 Hence, multiple tumour markers or screening for a super new marker can greatly improve the positive diagnostic rate for BC and reduce the negative diagnosis rate.
Over the next few years, imaging instruments still will be the routine method for screening BC, because they suit to be widely applied. However, new markers for BC will advance these technologies to higher throughput, faster, higher sensitivity and specificity. In the future, with development and use of these techniques, they not only can diagnose BC from various aspects, but also can evaluate effect of treating BC. Of course, different types of BC also will be evaluated by corresponding diagnostic methods, to get the most accurate results.

ACK N OWLED G EM ENTS
This work was funded by grants from the National Science

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
The article was written by Ziyu He and Zhu Chen. Miduo Tan

DATA AVA I L A B I L I T Y S TAT E M E N T
Some or all data, models or code generated or used during the study are available from the corresponding author by request.