All these technologies show amazing promise and some of them are already in use. The scientific community in India is constantly pushing their limits to get to a stage where the diagnosis of cancer will not be a life-altering event for patients
In the last few decades, cancer has become a leading cause of mortality worldwide. According to the WHO, currently, around 10 million new cancers are diagnosed each year worldwide, but unless there is an effective prevention campaign, the number will rise to 20 million in the next 17 years’ time. Therefore, the global scientific and healthcare community are turning to novel approaches in an attempt to make sure those grim projections don’t continue to haunt us. Stronger and effective cancer treatments are certainly part of the development goals, but a premium is also being put on early diagnosis to ensure better medical outcomes and assured prevention.
Today, more focus is given to precision medicine-quantitation, multiplexing and highly precise identification of markers. Precise tools which were once utilised in research settings are now applied in clinical practice with just one goal in mind- faster and more efficient cancer testing.
In this article, we examine top technologies that will improve efficiencies and precision in cancer diagnostics and prevention in India.
Fluid biopsies: Many scientific publications have documented that liquid or fluid biopsies are informative regarding response to a given therapy, are capable of detecting relapse with lead time compared to standard measures, and reveal mechanisms of resistance. According to Dr BS Ajaikumar, Chairman and CEO, HCG Global, liquid biopsy plays a significant role in those cases where it is difficult to establish a tissue diagnosis in a recurrent or metastatic setting. It is a simple minimally invasive procedure done on blood, plasma or urine sample to identify the genetic material of tumour cells either as ctDNA (Circulating tumour DNA) or CTC (Circulating Tumour cells) or cfDNA (Cell-free DNA) by identification of cancer-associated DNA / RNA / exosomes.
He further lists down some benefits of the same:
- It predicts response to targeted therapy with mutational load and by identifying specific, existing as well as new mutations. It is already in use for choosing targeted drug therapies in advanced stages of malignancies as in non-small-cell lung carcinoma (NSCLC). The US FDA approved the first liquid biopsy test in 2016 in NSCLC as a companion diagnostics test for exon 19 and exon 21 in EGFR, which is seen in 10-20 per cent of patients. About 60 per cent of these patients with known mutations (deletions), when followed up, are likely to develop additional mutations such as EGFR T790M indicative of resistance to the targeted drug therapy. So, the treatment can be modified accordingly with Osimertinib, even before the clinical evidence of failure to respond is evidence. Such detection is possible by liquid biopsy.
- It indicates prognosis through quantification of the ctDNA and residual mutational load as well as the type of mutation. It helps in detecting residual or recurrence or relapse of disease even in radiologically negative cases.
- Trials are underway to apply this technology for identification of mutations and targeted drug therapies in KRAS/NRAS in colon cancer, PIK3CA and resistant ALK in NSCLC. It has a role in the detection of clonal evolution and drug responses to targeted agents, especially by following mutations such as KRAS, NRAS, BRAF, TP53 and PIK3CA.
- Liquid biopsy using ctDNA is very useful for targeted therapy, follow up of these patients while on treatment and for early detection of recurrence/metastasis.
- Some trials have also confirmed the prognostic significance of CTCs in metastatic breast cancer. Studies are underway to evaluate the role of CTCs in identifying hormone-positive metastatic breast cancer patients who would benefit from early chemotherapy in comparison to hormonal therapy.
Real-time cancer diagnostics: With the need to need to translate recent discoveries in oncology research into clinical practice, cancer experts believe that objective, robust and cost-effective molecular techniques for clinical trials and, eventually, routine use is a must. Real-time PCR has become a useful and cost-effective technique for tumour profiling among clinical laboratories.
Dr Kirti Chadha, Head of Laboratory at Metropolis Healthcare expounds, “The pathogenesis of tumours is complex making the surgical management more difficult. Here comes the role of real-time diagnostics which will give an on-table diagnosis to make the treatment successful. A lot of research is going on it like an intelligent surgical knife has been developed by using an old technology where an electrical current heats tissue to make incisions with minimal blood loss, but with this technology, the vapourised smoke is analysed by a mass spectrometer to detect the chemicals in the biological sample allowing identification of malignant tissue. Also, a robotic platform has been developed in treating lung cancer. It combines robotics, software, data science and endoscope innovation to help diagnose lung cancer at an early stage with more accuracy and a lower risk of complications. Similarly, real‐time detection of breast cancer at the cellular level by a multispectral confocal scanning system has been developed. These are at research levels or some of them are being approved for use. Introducing advanced technology to traditional methods can also give us better real-time solutions like using digital pathology.”
Digital PCR: Digital PCR is the latest and advanced iteration of a conventional quantitative RT-PCR for sensitive and accurate measurement of DNA/RNA from samples. The primary principle behind the technique is similar to q-PCR but differs in the way the sample target is analysed.
Dr Dheeraj Gautam, Head of Department – Department of Histopathology, Associate Director- Department of Pathology and Lab Medicine, Medanta- The Medicity says, “PCR is a common test used to make many copies (millions or billions) of a particular region of DNA. With best systems, we have the capability to detect as few as ~10 copies of DNA templates. It is routinely used in DNA cloning, cancer diagnostics, and forensic analysis of DNA. For example, it might be a DNA sequence (gene) from a crime scene to match crime suspect, by forensic scientists. Typically, the goal of PCR is to amplify enough of the target DNA region, so that it can be analysed to deliver useful scientific information. Presently, Coronavirus is being tested by this method.”
Adding to this, Dr Ajaikumar informs, “Digital PCR is a simple and reproducible technique that does not rely on a calibration curve for sample target quantification. Digital PCR works by partitioning a sample of DNA into many individual, parallel PCR reactions. Following PCR amplification, the number of positive vs negative reactions is determined and the absolute quantification of target calculated using Poisson statistics. The benefits are, high precision, better signal to noise ratio, removal of PCR efficiency bias and simplified quantification.”
Speaking about the areas in which Digital PCR is currently applied, Dr Chadha reveals, “dPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. The form of digital PCR ie. Digital droplet PCR(ddPCR) is performed in Metropolis for circulating tumour DNA(ctDNA), EGFR/KRAS/NRAS/BRAF mutations in lung and colorectal cancer.”
Chromosome Analysis: Since altered genetic mechanisms lead to the development of cancer, chromosome analysis plays a significant role in the diagnosis and treatment monitoring of patients with various types of cancer. Chromosome analysis can be done by karyotyping and CGH(Comparative Genomic Hybridisation) array.
Dr Ajaikumar explains the various techniques that follow under chromosome analysis:
Cytogenetics(Karyotyping and FISH): FISH can identify chromosomal abnormalities such as insertions, deletions, translocations and amplification, through the use of fluorescent dyes that bind to sequences of interest. It is well known that certain types of cancer have specific genetic alterations. So far > 200 rearrangements and fusions have been identified. Examples include BCR-ABL translocation in CML, ALK rearrangement in NSCLC, Her-2 in Breast Cancer, Synovial sarcoma with t(X:18) (p11.2;q11.2), Ewing’s Sarcoma with t(11:22) (q24;q12.2). FISH is applied to detect genetic abnormalities that include different characteristic gene fusions or the presence of an abnormal number of chromosomes in a cell or loss of a chromosomal region or a whole chromosome. It is also applied in different research applications, such as gene mapping or the identification of novel oncogenes. FISH has high sensitivity and specificity. With microfluidics FISH, it can be faster and less costly.
Immunohistochemistry: Provides a platform for identification of certain chromosomal alterations through detection of proteins. Examples are the Her-2 testing in Breast and gastric cancer, ALK in NSCLC, TFE3 in Alveolar Soft Part Sarcoma, MDM2 and CDK4 in certain soft tissue sarcomas like Liposarcomas. They have high sensitivity and specificity of almost 95-97 per cent. They also provide targets for drug therapy.
Molecular testing: Through PCR, Direct sequencing, DNA and Protein microarray techniques and the latest path-breaking technology of NGS. PCR and sequencing are useful when we want to look for the presence of a known genetic alteration such as EGFR mutation in lung adenocarcinoma. But when the alterations are unknown or are likely to involve many loci, a panel of genetic markers can be screened through Next-Generation sequencing.
Next-Generation Sequencing (NGS): Massively parallel deep sequencing of a large number of patients with a variety of cancers to analyse the mutation profile of tumours at ‘one go’ provides a comprehensive understanding of the processes that drive an individual’s cancer. This will break the cycle of ‘trial and error’ medicine, and link the test to patient-tailored action and evidence-based therapy/ treatment plan in cancer. Furthermore, using genomic markers as response predictors to chemotherapy will dramatically improve response rates impacting the risk-benefit ratio for these patients.
Cell Signaling Pathway Testing: According to experts, cancer is caused by genetic and/or epigenetic changes in one cell or a group of cells. These alterations disrupt normal cell function and cause cancerous cells to proliferate and avoid mechanisms that would typically control their growth, division, and migration. Many of these disruptions map to specific cell signalling pathways. These pathways are involved in deregulated cell survival, cell differentiation and apoptosis. They form the ‘hallmark of cancer’ that include immune evasion, replicative immortality, activate invasion and metastasis, induce angiogenesis, resist cell death, deregulate cellular energetics, sustained proliferative signalling, evading growth suppressors, possess genome instability and mutations, and mediate a tumour associated inflammatory response. Signalling pathways such as Ras proteins through Raf-MEK-Extracellular signal-regulated kinase (ERK) and PI3K-AKT-mTOR pathways regulate cell survival, cell proliferation and migration/invasion in response to matrix adhesion and growth factor stimulation. Three Ras proteins – KRAS, NRAS and HRAS become mutationally activated and promote oncogenesis. Also identified are Wnt/Beta Catenin signalling in APC and NF2 gene in Neurofibromatosis Type 2. Certain growth factors such as RTK-VEGF, TGF Beta, PTEN in several types of cancers, are detected with either FISH or IHC or ELISA or other molecular profiling such as NGS more accurately and form the target for drug therapy.
Tissue microarrays: Tissue microarray technology overcomes the bottleneck of traditional tissue analysis and allows it to catch up with the rapid advances in lead discovery. Dr Chadha further explains, “Tissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. It is a recent innovation in the field of pathology. A microarray contains many small representative tissue samples from hundreds of different cases assembled on a single histological slide, and therefore allows high throughput analysis of multiple specimens at the same time. It can permit simultaneous analysis of molecular targets at the DNA, mRNA, and protein levels under identical, standardised conditions on a single glass slide, and also provide maximal preservation and use of limited and irreplaceable archival tissue samples. This versatile technique, in which data analysis is automated facilitates retrospective and prospective human tissue studies. It is a practical and effective tool for high-throughput molecular analysis of tissues that is helping to identify new diagnostic and prognostic markers and targets in human cancers and has a range of potential applications in basic research, prognostic oncology and drug discovery. This technique is very versatile as many downstream molecular assays such as immunohistochemistry, cytogenetic studies, Fluorescent In situ-Hybridisation (FISH) etc., can be carried out on a single slide with multiple numbers of samples.”
Adding further, Dr Ajaikumar points out, “The field of biomarker research can further be escalated by the integration of TMA technology with digital pathology. The most important disadvantage of this technique is that one small tissue core may not be representative of the whole tumour analysed conventionally. Therefore, many such cores of the same may be required to carry out analysis to arrive at a definitive conclusion. This is mainly significant for heterogeneous tumours like a human ovarian tumour. Whether this technology is useful in heterogeneous tumours is still highly debated.”
Artificial intelligence-based therapy: Many cancer care experts believe that integration of AI technology can improve the accuracy and speed of diagnosis, aid clinical decision-making, and lead to better health outcomes. AI-guided clinical care has, therefore, the potential to play an important role in reducing health disparities, particularly in low-resource settings. Dr Gautam exemplifies how AI integration can be done in cancer diagnosis. “A pathology AI system is a computer programme that assists pathologists in their work or provides automated pathology. Machine learning allows learning a task from data, like providing a diagnosis or a score, or a subtask, like classifying different cancers. A deep learning network is able to learn highly complex visual features just from the image data, achieving expert human performance,” he describes.
Similarly, citing more examples, Dr Chadha spells out, “AI also has gained importance in therapy designs, for e.g. Google is collaborating with health delivery networks to build prediction models from big data to warn clinicians of high-risk conditions, such as sepsis and heart failure. A neural network was applied to identify breast cancer with the inputs from mammographic images. A convolutional neural network was also performed to identify skin cancer from clinical images. Many start-ups are developing AI-derived image interpretation algorithms and can identify the patients at most risk as well as those likely to respond to treatment protocols. Digital pathology is one such platform which can be used for AI interpretations and diagnosis. Metropolis is one of the first CAP & NABL approved labs in India to adopt this platform. Scanning of slides to create a database which can be used for machine learning and AI-derived image interpretation.
On the anvil
As the scientific community continues to move ahead there will be cross-fertilisation of tests and technologies in the future. Oncologists and histopathologists also indicate a growing significance of pharmacodiagnostics in cancer care due to the development of new improved targeted drugs.
Adds Dr Chadha, “This emerging and expanding speciality with major potential for the specific linking of a treatment outcome like a response, toxicity and resistance to a key molecular alteration (e.g. protein overexpression or gene amplification) within a disease state to predict therapeutic response. It is used in measuring response or adverse side effects of both established and newer therapies. In oncology, recent advances with targeted therapeutics have demonstrated the critical importance of appropriate pharmacodiagnostic approaches. It is based on identifying somatic molecular changes in the tumour which forms the basis of molecular targeting of many novel therapies. The development of Herceptin (targeting the human epidermal growth factor receptor (HER2) oncogene in breast cancer) and Glivec (targeting BCR-ABL translocation in leukaemia) are excellent examples of the close relationship between target expression, pharmacodiagnostic tests and clinical therapeutic response. As treatment response depends on the molecular profile of individual cancer, the major challenge for the future will be to co-develop novel targeted therapies and pharmacodiagnostic tests also called as companion diagnostic tests that will predict patient response to therapy. To successfully integrate novel pharmacodiagnostics into clinical practice the collaboration between pharmaceutical and diagnostic industries, clinical oncologists and researchers must be strengthened.”
Going forward, experts reveal immense opportunities for IVD companies to thrive in this business segment. But to be successful, IVD companies must demonstrate the impact their technologies will have on physician’s decisions and patient outcomes.
raelene.kambli@expressindia.com
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