Microarray-Based Gene Profiling – A Promising Future For Cancer Diagnostics

Gene expression technology involving microarrays for cancer detection is forming the groundwork for many cancer detection researches thus to identifying new therapeutic targets.

Several innovative developments have been generated for the diagnosis and treatment of cancer over the past decade. A number of new approaches for analyzing alterations at both gene and protein level are being introduced for research and routine laboratory practice. Novel tools are available to ease the interpretation of huge sets of data within a short period of time. Use of microarray technology is one such tool which is revolutionizing the clinico-pathological research setting for cancer. Nearly 15 years have passed since the possibility of analyzing nucleic acid analytes in a massively parallel fashion was proposed using the then new concept of microarrays. A decade ago, proof of principle demonstration projects established the use of high-density microarrays to genotype multiple polymorphisms within a large gene, cystic fibrosis transmembrane regulator (CFTR), to rapidly analyze DNA sequences by hybridization and to ascertain differential gene expression of the entire genome of an organism. The use of microarrays has had an explosive influence on the rate at which new biological information can be learned, including in a non-hypothesis driven manner.

The past decade has also seen these research tools applied increasingly to questions of clinical and medical relevance. Genotyping drug metabolizing enzyme genes, resequencing important tumor suppressor genes, and classifying neoplastic disease by differential gene expression profiles are but a few of the many possibilities to provide clinically useful information using microarray-based diagnostic tests. The potential applications of this tool are highly increasing beyond horizons and are becoming popular amongst researches, pathologists, and basic scientists. Microarrays are now being classified as a high-throughput screening system with the ability to assay large amounts of biological material on a small 2D surface. The throughput capabilities of microarrays along with their small size are making them an exciting technology within biological research especially in cancer research. DNA microarrays, for example, have proven very useful in disease research, allowing for the interactions of hundreds to thousands of genes to be documented at one time. In conjunction with knowledge gained from sequencing the human genome, many microarray assays are being developed to allow profiling of the expression of thousands of genes. Microarray analysis of entire transcriptomes of clinical samples is extremely useful for understanding the molecular biology of disease, providing opportunities for molecular classification of disease, the identification of novel molecular target for intervention of disease, and the prediction of therapeutic response.

According to WHO’s recent data, cancer is the second leading cause of death globally, and will be responsible for an estimated 9.6 million deaths in 2018. Globally, about one in six deaths is due to cancer. Early observation of this disease is needed to prevent further growth. Moreover, cancer detection is also important since cancer mortality can be reduced if cases are detected and treated early. Gene expression technology involving microarray technology for cancer detection is nowadays forming the groundwork for many cancer detection researches. The new molecular profiling has enriched the understanding of cancer heterogeneity and yielded new prognostic and predictive information. The molecular signatures so identified help not only in revealing the biological spectrum of cancers, but simultaneously also provide diagnostic as well as prognostic and predictive gene signatures, and may identify new therapeutic targets. Microarray profiling has, unquestionably, been now established as a powerful tool in unraveling mechanistic insights into tumor biology. It is believed that this is one of the greatest achievements in the field of science and will revolutionize and remold biology.

Indian dynamics
The burden of cancer in India is increasing day by day. According to projections of the Indian Council of Medical Research (ICMR), the total number of new cancer cases is expected to touch 17.3 lakh by 2020. The number of deaths due to cancer is estimated to likely reach 8.8 lakh cases by 2020. The data regarding cancer in India show that only 12.5 percent of patients come for treatment in the early stages of the disease. This clearly indicates the need for greater awareness and early detection. The central government supplements the efforts of the state government for improving healthcare including prevention, diagnosis, and treatment of cancer. The objectives of National Program for Prevention and Control of Cancer, Diabetes, Cardiovascular Diseases and Stroke (NPCDCS) being implemented under National Health Mission (NHM) for interventions up to district level include awareness generation for cancer prevention, opportunistic screening, early detection, and referral to an appropriate level institution for treatment. A population-level initiative of prevention, control, and screening for common NCDs has been rolled out in over 150 districts of the country in 2017-18 under NHM, as a part of comprehensive primary healthcare. The screening activity will generate awareness on risk factors of common NCDs including cancer.

The Government of India is also implementing Strengthening of Tertiary Care for Cancer Facilities scheme under NPCDCS to assist to establish/set up state cancer institutes (SCI) and tertiary care cancer centers (TCCC) in different parts of the country. Oncology in its various aspects is the focus for newly set up AIIMS and many upgraded institutions under Pradhan Mantri Swasthya Suraksha Yojana (PMSSY). Setting up of the National Cancer Institute at Jhajjar, Haryana and a second campus of Chittranjan National Cancer Institute, Kolkata has also been approved by the government. These institutes will be apex institutes for activities related to cancer including research and treatment. Apart from initiating measures to establish oncology wings in various medical colleges, the central government has upgraded several regional cancer centers into SCIs by providing adequate funds to build infrastructure, procure state-of-the-art equipment for diagnosis and treatment of cancer. One of the beneficiaries of this national initiative is Karnataka, which has successfully upgraded the cancer care facilities at Kidwai Cancer Institute. All these will enhance the capacity for cancer research in the country leading to increased adoption of microarray technologies.

Having knowledge about molecular events of cancer can be very helpful in choosing an effective treatment and its results can be more effective and accurate compared to conventional methods of cancer detection and treatment. Microarray technology provides the possibility of examining the tumor behavior in the living tissue and drug resistance of the patient. No doubt, there is a need for making increased investments in the area of cancer prevention research. The country needs to establish cancer units for early detection, diagnosis, treatment, and to provide palliative care in rural areas where about 70 percent of the population resides. North eastern states have been lagging behind in cancer research and need infrastructure development to promote research in these states. The tripartite MoU signed between Barooah Cancer Institute Guwahati, Assam Government, and the North-Eastern Council, as a result of which, super-specialty cancer courses are being started for the first time in the northeast region, is a step in the right direction to initiate cancer research.

Global market dynamics
The global microarray market is expected to grow at a CAGR 14.5 percent from 2018 to 2024, according to Research and Markets. The market is majorly driven by the increasing prevalence of cancer and rising need of research in pharmaceutical industries, and growing application areas of microarrays. Increasing prevalence of different chronic diseases, use of this techniques for the diagnosis of infectious diseases, development in the field of genetics, and increasing healthcare expenditure; all these together have provided a push for this market’s growth. However, high cost of tests and need of high tech instruments may restrain growth of this market.

In 2018, the consumables segment is expected to account for the largest share of the global market. The large share of this market segment can be attributed to the growing applications of microarray in various fields and the regular, repeated, and bulk purchases of consumables. The DNA microarrays segment, in 2018, is expected to account for the largest share due to the use of DNA microarrays in various applications such as drug discovery, genomic and cancer research, personalized medicine, and genetic disease diagnosis. Moreover, based on the application, the drug discovery segment is estimated to register the highest CAGR. Microarrays have multiple applications in drug discovery as they help to measure the expression patterns of thousands of genes to identify appropriate targets for therapeutic intervention. Microarrays are also an integral part of therapeutic drug discovery, optimization, and clinical validation as they help to prioritize a few genes as potential therapeutic targets.

In 2018, North America is expected to account for the largest share of the global market owing to the presence of a well-developed healthcare sector, increasing incidence of cancer and genetic diseases along with government support for funding made available for genomics and proteomics research and development. Europe is the second largest market and is followed by Asia-Pacific. The presence of a large cancer population and increasing need for better diagnostic treatment have driven the European market. The Asia-Pacific microarray market is majorly occupied by Japan. India and China are the growing markets for microarray instruments and reagents.

Among many layers, some of the key players in the market are Qiagen, Thermo Fisher Scientific, Agilent Technologies, Applied Micro Arrays, Merck Sharp & Dohme Corp, Molecular Devices, Bio-Rad Laboratories, PerkinElmer, Illumina, Arrayit Corporation, BioGenex, and GE Healthcare. 

Scope for further improvement
The greatest challenge in using this technology is its expense. Making microarray cost efficient by developing the related software and robotics technology further, can make it more reliable in any field. Enhancing reproducibility and reliability of the data accessed through this method can also boost the applications of the microarray. Combining DNA microarray with proteomics can help in understanding the way in which pathogens react in the microenvironment. A detailed study of these reactions can help in the identification of bacterial virulence factors, and aid in the design of new vaccines. This young technology has the potential to create opportunities for scientists to discover drugs at a faster rate and shall open horizons to study various gene and protein expression patterns in a wide range of normal, cancerous, and non-cancerous tissues. Further researches in this area can lead to the discovery of personalized medicines depending upon the genetic buildup of the patients. This technique can be supplemented with automation which shall further reduce the burden on the personnel and generate more precise information. Archival of many scarce tissues on a single block and digitization of the information can help in conservation of huge data sets for decades which may be useful to carry retrospective analysis of novel markers.

Microarrays have the potential of multiplexed detection, but they are not portable (due to heavy and expensive scanners) and additionally require long incubation (with the sample) time. However protein microarrays, and particularly antibody microarrays (or immunoarrays), have the potential of becoming portable devices capable of detecting multiple biological targets, thus serving as ideal diagnostics and monitoring tools. A number of characteristics of the present methods are holding back the realization of this potential such as large dimensions of the active sites (spots) that result in the requirement of scanners, thus hindering the portability of the technology; lack of integration of the microarray with detection, sample (liquid) handling, read-out, power and remote reporting systems preventing autonomous operation (requiring human handling). The present technology allows automated manufacturing of spots with ~100 m diameter, with a center-to-center spacing of ~300–400 m. Miniaturization of the spots, without sacrificing the signal-to-noise ratio has been a long time goal. Many efforts are being made to miniaturize protein microarray spots. The most common methods include nano grafting, dip-pen nanolithography, and nano-fountain pen (NFP). The incubation time can be controlled and diffusion distances shortened by reducing the dimensions of the system. Furthermore, equilibration can be accelerated by flowing the target molecules continuously thereby enhancing the mass transport of the target molecule. Thus, integration of the miniaturized microarray with microfluidics will lead to shorter incubation periods, in addition to the increased convenience of sample handling.

Ongoing research to develop more efficient microarrays
3D printing has gained popularity in recent years as a means for creating a variety of functional products, from tools to clothing and medical devices. Now, the concept of multi-dimensional printing has helped a team of researchers at the Advanced Science Research Center (ASRC) at the Graduate Center of the City University of New York develop a new, potentially more efficient and cost-effective method for preparing microarrays. Researchers with the ASRC’s nanoscience initiative have combined microfluidic techniques with beam-pen lithography and photochemical surface reactions to devise a new biochip (microarray) printing technique. The process allows scientists to repeatedly expose a single chip to the same or different factors and imprint the reactions onto different sections of the biochip. The result is a biochip that can accommodate more probes than is achievable with current commercial platforms.

An additional benefit of the new tool is that it allows researchers to reliably print on a variety of delicate materials – including glasses, metals, and lipids – on the length scale of biological interactions, and without the use of a clean room. It also allows scientists to fit more reactive probes onto a single chip. These improvements could, in theory, reduce the cost of biochip-facilitated research. ASRC scientists are now exploring ways to fine tune their new technique for creating these microarrays. They want to be able to record even more complex surface interactions and reduce our resolution down to a single molecule. This technique gives rise to a new method of microarray creation that should be useful to the entire field of biological ‘omics research.

The path ahead
Accurate and early diagnosis of cancer is vital. For this reason, using microarray technology to recognize the disease is getting popular. It is likely that microarray-based gene expression profiling will continue to play a central role in understanding the molecular biology of cancer, identification of new therapeutic targets, prognosis of disease outcome, and therapy response. Continual improvements to the technology and data analysis methods will also allow these approaches to be developed as in vitro diagnostic tests. To date, virtually all microarray tests based on differential gene expression have been applied in pure research settings; development of this promising technology into robust diagnostic applications will require much additional effort. Validation of diagnostic applications of gene expression microarray technology will require well-designed, controlled clinical studies. Microarray technology is complex and requires many steps throughout the process from obtaining the clinical sample to data analysis. In order to develop assays that exhibit robust performance acceptable for a diagnostic test, it is imperative to define well-documented and standardized assay protocols. Ideally many quality control steps will be embedded in the process that will serve as a gatekeeper preventing diagnostic error.

Since its introduction, the microarray platform has experienced a tremendous growth and currently it is a powerful tool used in various biological applications. However, it has not been embraced in the molecular diagnostic market as much as it was anticipated in the early days. The challenge comes from the skepticism on the reproducibility of the microarray data and on the reliability of the biological interpretations inferred. In addition, the microarray technology is suffering from strong competitions by other bio-diagnostic techniques. On the other hand, integration of various steps of the microarray assay into a harmonized and miniaturized handheld device suitable for point-of-care (PoC) has been a goal for the microarray community. In this respect, significant progress has been achieved in coupling the DNA microarrays with efficient liquid manipulation microsystems as well as developing novel technologies of supporting subsystems that well suit future PoC microarray devices. While today there are commercial microarray options with greater sensitivity, the simple production of microarrays has allowed for easily customizable experiments within laboratories. For labs currently equipped with microarray instrumentation and with established operational workflows for sample processing and data interpretation, microarray methods will likely remain competitive for some years to come.