Next-Generation Sequencing can revolutionize cancer care in Bangladesh

 

Bangladesh has achieved a significant reduction in mortality from infectious diseases but shows a recent increase in cancer morbidity.¹ Cancer awareness is low among the population, and screening programs are vastly insufficient. Due to a lack of proper diagnosis, most cancers get the chance to develop to advanced stages, rendering them difficult to treat. The average 5-year survival rate of cancer patients is 91% if detected at earlier stages and drops to 26% if diagnosed at later stages.² Cancer is a genetic disease, meaning that it develops due to alterations in genes.³ Although Bangladesh has various infrastructures and institutions, there is inadequacy in genetic testing of cancers. Next-generation sequencing can produce extensive genomic information in a short time,⁴ analysis of which can reveal disease markers. Integration of next-generation sequencing in cancer diagnosis will lead to rapid and appropriate results, prompting effective treatment.

In Bangladesh, estimates show a more than 70% mortality rate in all the diagnosed cancer cases.¹ This dismal situation arises mainly due to improper or late diagnosis and partly due to non-specific treatment. Apart from a handful of people living in the city, most of the population bear a significant risk of late diagnosis due to a shortage of cancer specialists in the country.¹ Conventional cancer diagnosis in the country starts with imaging of suspicious organs, followed by a biopsy, where extracted tissue samples undergo critical analysis under the microscope. Disease detection in this manner has pitfalls like late detection and plausible misinterpretation. In Bangladesh particularly, the facilities' unavailability poses an additional constraint. However, genomic technologies show tremendous promise in improving early detection. Before comprehending their usefulness, it is necessary to understand cancer development first.

Within each of the trillions of cells in the body, there are long molecules called DNA, which chemically store all hereditary information. The complete set of this information is the cell’s genome and is subject to changes, or mutations, throughout their lifetime.³ These mutations produce diversity among the cells and may confer advantageous characteristics to some of them. If a cell can acquire enough such mutations to enable it to, among other things, proliferate uncontrollably, evade programmed death, and invade tissues, then it can become cancerous.⁵ With time, a cancer cell can evolve to escape the scrutiny of the immune cells and become more aggressive. If cancer cells turn out to be successful in migration to various parts of the body, they can cause recurrence even after the removal of their original organ.⁵ That’s why cancer detection in its rudimentary stages is a must to improve the treatment efficacy. Early detection is also proven to elongate survival time by several years.⁶

The mutations in cancerous cells can be revealed through DNA sequencing. Since cancers accumulate mutations throughout their development, mutational analysis can decisively detect real-time cancer characteristics.³ Prior to the emergence of next-generation sequencing, genome sequencing was a tedious process as it took 11 years to sequence the first human genome.⁴ Next-generation sequencing, or NGS for short, can now sequence an entire human genome in just three days.⁴ The process begins with the isolation of the DNA content from cells. The DNA molecules are then fragmented into thousands to millions of pieces and modified according to the NGS platform.⁴ These modifications guide the fragments to adhere to special structures in the sequencing apparatus. Finally, sequencing and subsequent analysis of these fragments lead to a complete genomic profile. Since NGS involves numerous simultaneous sequencing reactions, it is also called massively parallel sequencing. Although several NGS methods are available, these are broadly categorized either as second-generation or third-generation sequencing.⁴ Second-generation sequencing uses relatively short DNA fragments. Adequate multiplication of the fragments precedes sequencing and has relatively lower error rates.⁴ However, because of their use of short-reads, special genomic features like repetitive DNA cannot be resolved efficiently.⁴ On the other hand, third-generation sequencing uses long reads of single DNA molecules. Although it results in faster sequencing and resolution of a wide range of genomic properties, these benefits come at the cost of relatively higher error rates. Incorporating second-and third-generation sequencing in a hybrid manner can compensate for both of their shortcomings.⁴ The level and nature of RNA molecules, and DNA methylation pattern give an idea of the active genes in a cell.⁶ Apart from sequencing DNA bases, NGS technologies can also reveal the identity of these RNA molecules and methylation patterns.^4,6 Such information presents a profound picture of the functional status of any cell.

The analysis of data comes after the sequencing step. Bioinformatics software programs are utilized to find aberrations in the genome. They can perform computational analyses to search for mutations in patient’s DNA by comparing them with those curated in databases. Finally, if analogous mutational signatures are found, details of the cancer type and its characteristics can be disclosed.  The International Cancer Genome Consortium (ICGC) and the Catalogue Of Somatic Mutations In Cancer, or COSMIC for short, are two of the most prominent databases that store mutational information of human cancers. COSMIC for example contains 6 million mutational data from 1.4 million tumour samples.⁷ These databases are constructed on genomic data sequenced using NGS. Two main projects that sequenced an enormous amount of individual cancer genomes are the Cancer Genome Project and the Cancer Genome Atlas.⁷ Their contribution reveals the wide spectrum of mutational signatures that cancers can have. For example, ultraviolet exposure gives different mutations than tobacco smoke.³ Again, recurrent cancers have drug-resistant mutations not seen in early-stage cancers.³ However, because of the lack of genomic data from Bangladeshi cancer patients, mutational signature analysis is yet to be fully optimized for this region. Usage of NGS can solve this problem through widespread and repeated monitoring of cancers as they evolve.

Because of next-generation sequencing, less-invasive techniques like blood tests can now be utilized for genomic analyses. Many traditional blood tests are already in clinical use to assist cancer diagnosis. For example, excess white blood cell count indicates leukemia, an elevated level of prostate-specific antigen points to prostate cancer.² Unfortunately, these tests are not conclusive, and many tumours don’t produce detectable chemical markers at early stages. However, advancements in molecular biology techniques can now isolate tiny amounts of circulating tumour cells (CTCs) and their DNA (ctDNA) that are sloughed off into the bloodstream.⁶ Since these DNA fragments are usually present in small amounts, ample amplification is required before sequencing can take place.⁶ Liquid biopsy tests subject the ctDNA to second-generation sequencing, and bioinformatics analyses can help uncover if abnormal cells are growing in the body.⁶ Furthermore, since ctDNA level increases with tumour size,⁶ those may also be used for cancer staging. Liquid biopsy shows the potential to detect cancer growth years before conventional diagnosis.⁶

However, when there is a meager amount of DNA, or missing parts of the genome typically found in ctDNA; whole-genome sequencing is not applicable, and different approaches need to compensate. Cancer genome analyses have revealed that several mutations are fairly common to many cancers, and many more mutations are restricted to particular cancer types. These common mutations are known as driver mutations.³ Driver mutations are usually clustered in subsets of genes; rather than randomly distributed.³ For these reasons, it is feasible to selectively analyze those genes that are most likely to be mutated under specific disease conditions. Customized gene panels in second-generation sequencing platforms can selectively amplify genomic segments of particular interest, increasing sensitivity for those regions of interest in the ctDNA.⁶ One NGS setting called Cancer Personalized Profiling by deep sequencing (CAPP-seq) captures distinct DNA sequences and showed 10,000x coverage of DNA containing high driver mutations. It means that particular ctDNAs can be detected even if it comprises only 0.01% of all the DNA molecules in a sample.⁸ Recently, considering protein markers with ctDNA has resulted in even higher detection sensitivity of cancers at early stages.⁸

However, diagnosis is not the only area where next-generation sequencing and computational data analysis can contribute. In the early stages, cancers generally stay within a particular region in the body. In these cases, localized treatments such as surgery and radiotherapy become fruitful. Chemotherapies target all the rapidly dividing cells of the body and effectively destroy cancer cells if resistance against those drugs does not develop. However, if cancer cells can survive these assaults, they reach later stages and start invading surrounding tissues. Eventually, their settlement in other parts of the body results in metastatic cancer.⁵ Since metastatic cancer cells usually grow alongside healthy cells of major organs, eliminating only the malevolent ones become essential. This is where precision medicine can play a major role. Novel treatment strategies like targeted therapy and immunotherapy select only the susceptible cells for elimination and thus show promise to tackle metastatic cancers.

Cancer cells’ metabolic activities are somewhat different than normal cells, and targeted therapies exploit these differences for their selective elimination. Knowledge of the mutated genes plays a pivotal role in identifying these aberrations. There can be hundreds of distinct mutated genes and their respective treatment arms.⁹ Unfortunately, in Bangladesh, only three common hormone-dependency genes are screened in breast cancer tissues for targeted therapy. Even among the hormone-dependent cancers, only one-third of the patients can achieve full recovery. Its cause is a phenomenon referred to as tumor heterogeneity,⁹ where one drug cannot destroy all the cells in a tumour mass. Next-generation sequencing can search for thousands of mutable genes as well as reveal heterogenic subtypes within a tumour.⁹ Currently, tumour samples of Bangladeshi patients are sent to India for comprehensive genomic analysis, which consumes valuable time. Installing NGS platforms throughout the nation’s hospitals can reduce this lag in treatment and provide personalized treatment regimens.

An emerging cancer treatment strategy is immunotherapy, which involves promoting the body’s immune system, especially T cells, to fight cancer.¹⁰ T cells constitute part of the body’s immune system and eliminate rogue cells. Evading the body’s immune surveillance is one of the most crucial hurdles that cancer needs to pass.⁵ Successful cancers manage to deceive the T cells and get treated as normal cells. Some cancers overexpress certain checkpoint molecules that prevent T cell activation. Checkpoint inhibitors thus can be immunotherapeutic drugs that let the T cells bypass the checkpoints in killing cancer.¹⁰ Cancer’s genomic profile can determine if and which checkpoint inhibitor is likely to be effective, for which NGS and bioinformatics again become practical tools.

Two other immunotherapies target mutated proteins called neoantigens exhibited on cancer cell surfaces.¹⁰ After synthesizing these neoantigens using recombinant technology, vaccines can be produced which adapt T cells to specifically target cancer cells.¹⁰ Furthermore, T cells can be genetically engineered to produce chimeric antigen receptors,¹⁰ which assist their binding to neoantigens, eventually destroying the cancer cells. Although yet to be widely applicable, appropriate immunotherapies so far have shown unprecedented curability. Since neoantigens are associated with mutations in the genes, DNA sequencing is the most convenient method to identify them, a feat NGS can overcome easily.

DNA being the hereditary material, can pass mutations to offspring, predisposing them to future cancer development. For example, mutations in the BRCA1 and BRCA2 genes impose a lifetime risk of developing breast and ovarian cancers upon an individual.⁸ If patients contain such mutations, their family members are likely to have them too; rendering them a higher probability of cancer development. In such cases, NGS-based early screening programs like liquid biopsy can play a crucial role in reducing their cancer morbidity.

Next-generation sequencing can provide fast and accurate genomic information that can be beneficial not only for early disease detection but also for proper treatment. Advancements in software programs, databases, and biological equipment are pioneering new methods to integrate NGS in cancer care. Cancer morbidity in Bangladesh will significantly reduce if all the hospitals can attain next-generation sequencing capability.

 

 

 

 

References:

1. World Health Organization. Bangladesh. Cancer Country Profile 2020. https://www.who.int/cancer/country-profiles/BGD_2020.pdf (2020).

2. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer Statistics, 2018. CA Cancer J Clin 68, 7–30 (2018).

3. Stratton, M., Campbell, P. & Futreal, P. The cancer genome. Nature 458(7239), 719–724 (2009).

4. Kumar, K., Cowley, M. & Davis, R. Next-Generation Sequencing and Emerging Technologies. Semin Thromb Hemost 4, 661-673 (2019).

5. Hanahan, D. & Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell 144(5), 646-674 (2011).

6. Chen, M. & Zhao, H. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics 13, 34 (2019).

7.  Tate, J. et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res 47, D941-D947 (2018).

8. Postel, M., Roosen, A., Laurent-Puig, P., Taly, V. & Wang-Renault, S. Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective. Expert Rev Mol Diagn 18, 7-17 (2017).

9. Gu, G., Dustin, D. & Fuqua, S. Targeted therapy for breast cancer and molecular mechanisms of resistance to treatment. Curr Opin Pharmacol 31, 97-103 (2016).

10. Mukherjee, S. Genomics-Guided Immunotherapy for Precision Medicine in Cancer. Cancer Biother Radiopharm 34, 487-497 (2019)