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DNA Sequencing: Another Weapon Against Cancer

Gene mutations and dysregulation of cellular pathways lead to the creation of cancer cells and potentially to tumour formation. Usually, the genes involved in tumour formation are oncogenes and tumour suppressor genes. Most oncogenes begin as proto-oncogenes. Proto-oncogenes are normally regulating cell proliferation, growth and differentiation and they control cell cycle. If this proto-oncogene is mutated and consequently activated at a time-point that it should be silenced, then it becomes an oncogene. In this case, it starts to promote cell proliferation. On the other hand, tumour suppressor genes normally exist in the human body to slow down cell proliferation when needed. A mutation in a tumour suppressor gene may stop its action and cell proliferation can get out of control. Therefore, inactivation of tumour suppressor genes and hyperactivation of oncogenes can result in tumourigenesis. Even in the early stages, cancer is characterized by genetic instability, which causes malfunction of the regulatory pathways and accumulation of mutations in the patient’s genome. Visualizing and understanding the genetic changes that arise during cancer not only benefits the individual patients, but it also enhances current knowledge on the topic of cancer. New sequencing techniques and bioinformatics platforms have enabled efficient genetic analysis of tumour samples.


Background Knowledge On DNA Sequencing

DNA sequencing is a cutting-edge molecular technique that determines the precise order of nucleotides in a DNA molecule. By analyzing the sequence of these building blocks (adenine, cytosine, guanine and thymine), scientists can unravel the genetic information encoded within the DNA. This technology has transformed the field of genomics, enabling comprehensive study of genes, genetic variations and their roles in various biological processes, from health and disease to evolutionary history.


One of the most popular types of sequencing is Sanger sequencing. This is known as the “chain-termination method”. This method requires a DNA primer, a DNA polymerase, normal deoxynucleotide triphosphates (dNTPs), which are the DNA bases, and di-deoxynucleotide triphosphates (ddNTPs), which prevent the extension of the DNA chain. ddNTPs are labelled with a radioactive or fluorescent tag. DNA is firstly denatured in order for the double chain to separate. The primer is then able to anneal on the one strand of the template DNA. Four different reactions take place each time in four different tubes. Along with the DNA polymerase, each reaction vessel contains all four dNTPs and one of the four ddNTPs. The DNA polymerase adds dNTPs on the template, according to complementarity, until a ddNTP is added. Each time this happens, the reaction stops and DNA fragments of different lengths are formed in all tubes. Polyacrylamide Gel Electrophoreses allows the analysis of those reactions and the determination of the DNA sequence. Nowadays, due to its high-cost and low efficiency, Sanger sequencing is mostly used in low-throughput, short-read projects. However, this technique is still valued because of its simplicity and sensitivity (Pareek, Smoczynski, & Tretyn, 2011).

Figure 1: Sanger Sequencing Data (Smith, 2019)

Although Sanger sequencing has been developed over the years, new more high-throughput techniques have become popular. Next-generation sequencing (NGS) techniques enable the rapid and parallel sequencing of millions of DNA fragments simultaneously. NGS techniques for example can sequence the entire human genome in just one day. NGS represents a groundbreaking leap in genomic research and has revolutionized the field of molecular biology. This happens because millions of DNA fragments are being sequenced in parallel (Behjati & Tarpey, 2013). The four basic steps in NGS are 1) DNA or RNA isolation, 2) library preparation, 3) amplification and sequencing and 4) analysis of the sequencing data. NGS is used to rapidly sequence whole genomes, deeply sequence specific target regions, identify pathogens and discover and study epigenetic factors. Moreover, NGS is an incredible tool to study tumours, analyse variants and cancer-related mutations (Hu, Chitnis, Monos, & Dinh, 2021). This remarkable technology has significantly decreased both time and cost, making it accessible to a wider range of researchers and clinicians. As the power and versatility of NGS continue to expand, its impact on diverse fields, from diagnostics to agriculture, promises to shape a brighter future for scientific discovery and medical advancements.

Figure 2: NGS Data (Campbell, 2020)

Nowadays, there are numerous types of sequencing, including DNA sequencing, RNA sequencing, Nanopore sequencing, short-read sequencing, long-read sequencing etc. These techniques are used by almost all biochemical labs in the world, as vital parts of research.


Tumour Sequencing Benefits Cancer Patients

Tumour sequencing can offer an analytic view of all the genes involved in the development of cancer. Such genes could be mutated, hyperactivated or silenced, disrupting cellular stability. Consequently, it is possible to detect the pathways that the tumor cells of a particular patient have become dependent on. This knowledge enables the design of personalised treatment for the cancer patient. After detecting a particular mutation that is thought to promote tumourigenesis, gene therapy techniques can specifically target that mutation and correct it. Especially in early stages of cancer, this approach can prevent the accumulation of mutations and stop cancer spread. Personalised treatments target the overregulated pathways of the patient’s molecular environment. Specifically designed drugs can target the misregulated proteins of the pathway. Precise and personalised treatment not only is more efficient, but also eliminates the presence of undesirable side effects. Finally, individual patients can also know the risk of recurrence. After treating cancer, it is still possible for cancer cells to be reactivated and create a new tumor. Knowing the genetic background of the tumour, enables the provision of its progression.


Tumour sequencing can potentially enable the diagnosis of cancer in the early stages before growth and metastasis of the cancerous cells. Genetic mutations in cancer are either sporadic or hereditary. Cancer is usually characterized by sporadic mutations, which are random and non-hereditary. However, in the rare event of hereditary cancer, germline mutations are being passed to the next generations. Development of cancer in the offspring is not guaranteed, however the possibility of such an event is increased (Schon & Tischkowitz, 2017). Sequencing of patient tumor samples could predict the development of cancer in other family members and offer the opportunity of early diagnosis and treatment. Breast cancer is atypical example of a partially hereditary cancer. BRCA1 and BRCA2 gene mutations are responsible for about 25-50% of hereditary cancer cases. Women with BRCA mutations have therefore an increased likelihood of developing breast cancer (Zhu et al., 2016). Mastectomy is a cancer treatment technique based on removal of breast tissue of breast cancer patients or individuals with extremely high risk of developing it. Tumor sequencing would make individuals aware of their risk to develop cancer and give them the chance to prevent it.


Understanding Cancer Development and Progression After Tumor Sequencing

Next generation sequencing (NGS) facilitated the analysis of cancer genome in the past. The Cancer Genome Project and The Cancer Genome Atlas sequenced about 37,000 tumour samples, gathering data which are now available in online databases (Ganini et al., 2021). Such projects established that the majority of cancers are characterized by point mutations as well as small indels, structural instability and alterations in the copy number of genes and chromosomes. Moreover, it was found that although there are mutations in the coding regions, the majority of cancer-related mutations are intergenic. Further information was released after the conduction of the Pan-Cancer Analysis of Whole Genomes (PCAWG), a sequencing project that focused on cis-regulatory sites, non-coding RNAs and large-scale structural alterations. This project managed to identify mutation patterns within 2,600 cancer whole genomes, detect mutational and structural signatures (which were also found to be altered over time) and recognize driver mutations in the genomes (Giunta, 2021). The 100,000 Genomes Project, which is currently being conducted, analyses the genomes of patients with cancers and other rare diseases. This project aims at creating genetic profiles of the patients, linking them to clinical and patient records. A similar approach has been performed in order to specifically identify patterns of mutations associated with cancer related to tobacco smoking.


Future tumour sequencing projects will further expand the gained knowledge on cancer. Tumour sample sequencing could lead to the identification of tumour suppressor genes and oncogenes that have not been recognized before. Until now, tumour suppressor genes such as Rb, p53 and BRCA2 have been recognized and their role in cancer has been extensively studied (Swale & Quinn, 2000). Examples of known oncogenes are Ras, Myc and Cyclin D and they are all associated with multiple types of cancer. More genes could be possibly implicated in the development and progression of cancer. Identifying all cancer involved genes would give information about the generation of tumours and it would expand the potential drug targets to cure cancer.


Figure 3: DNA molecule (Green, 2019)

As mentioned before, after sequencing a wide range of tumour samples, it is possible to produce a mutation spectrum of alterations linked to cancer and observe patterns of mutations. This could lead to the detection of the source of the mutations and the recognition of the disrupted pathways. Furthermore, it would be plausible to study the effect of carcinogens on the body and identify the types of mutations that they induce.


Even more useful it would be to study the changes in genome over time during the development and the progression of a cancer. This would give insight into the events that lead to the increase of tumour growth and metastasis. In addition, understanding the mutational events in a tumour would explain the therapeutic resistance of some tumour types and would bring us closer to designing techniques to overcome it.


Most importantly tumour sequencing should be performed in a large number of samples in order to compare and contrast the mutational profiles of the patients. Comparing multiple cancer genomes to identify genes that are frequently mutated will allow the identification of driver mutations. It will also allow the organization of tumours in subtypes in reference to their genetic background. The more genomes sequences are compared, the greater the sensitivity and the accuracy of the obtained information.


It is important to mention that sequencing tumour samples would give insight into cancer metastasis. Primary and metastatic tumours probably have genetic differences (Suhail et al., 2019). Sequencing may reveal the mutations that drive metastasis and provide an advantage in a new microenvironment that promotes tumour growth. This kind of information could generate personalised treatments that prevent cancer expansion in the organism.


Finally, sequencing of tumour samples would enable the understanding of the heterogeneity within tumors. Conventional sequencing of tumour biopsies containing millions of cells produce a consensus sequence result, which does not give information on individual cells. Contemporary techniques use single samples, performing single cell sequencing. Although amplification of the genetic material, a highly error prone method, is required, the amount of obtained information is significantly higher. Single cell sequencing has illustrated the genetic heterogeneity within a tumour (Lei et al., 2021). Genetic instability in cancer promotes the formation of cell subpopulations. Each of the different cancer cells in the tumour has distinct properties. Drug treatments may target the majority of the cancerous cells and efficiently kill them. However, a small cell subpopulation which might be resistant to the drug, could potentially continue proliferating and lead to cancer recurrence. Knowledge of the cell subpopulations and their genetic characteristics could ameliorate cancer drug design and facilitate understanding of the diversity of cancer.


Conclusion

Sequencing techniques have emerged as indispensable tools in the study of cancer due to the disease’s complex and multifactorial nature. Despite significant progress in identifying cancer-related genes and pathways, fundamental questions surrounding tumour growth, metastasis, and drug resistance still persist. Tumour sequencing offers a promising avenue to unravel the intricate molecular background of cancer, shedding light on the sequence of events that culminate in this life-threatening ailment. By deciphering the genetic profiles of tumours, researchers can gain deeper insights into the disease’s heterogeneity, enabling more targeted and personalized therapeutic approaches. The knowledge garnered from sequencing analyses will facilitate the development of gene therapies that can precisely target cancer at its root, offering new hope and improved outcomes for patients battling cancer. As the field of sequencing continues to advance, it holds great potential to drive transformative advancements in cancer research and ultimately improve patient care.


Bibliographical References

Behjati, S., & Tarpey, P. S. (2013). What is next generation sequencing? Archives of Disease in Childhood - Education & Practice Edition, 98(6), 236–238. doi:10.1136/archdischild-2013-304340

http://dx.doi.org/10.1136/archdischild-2013-304340


Ganini, C., Amelio, I., Bertolo, R., Bove, P., Buonomo, O. C., Candi, E., … Melino, G. (2021). Global mapping of cancers: The cancer genome atlas and beyond. Molecular Oncology, 15(11), 2823–2840. doi:10.1002/1878-0261.13056

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Giunta, S. (2021). Decoding human cancer with whole genome sequencing: A review of PCAWG project studies published in February 2020. Cancer and Metastasis Reviews, 40(3), 909–924. doi:10.1007/s10555-021-09969-z

https://doi.org/10.1007/s10555-021-09969-z


Hu, T., Chitnis, N., Monos, D., & Dinh, A. (2021). Next-generation sequencing technologies: An overview. Human Immunology, 82(11), 801–811. doi:10.1016/j.humimm.2021.02.012

https://doi.org/10.1016/j.humimm.2021.02.012


Lei, Y., Tang, R., Xu, J., Wang, W., Zhang, B., Liu, J., … Shi, S. (2021). Applications of single-cell sequencing in cancer research: Progress and perspectives. Journal of Hematology & Oncology, 14(1). doi:10.1186/s13045-021-01105-2

https://doi.org/10.1186/s13045-021-01105-2


Pareek, C. S., Smoczynski, R., & Tretyn, A. (2011). Sequencing Technologies and genome sequencing. Journal of Applied Genetics, 52(4), 413–435. doi:10.1007/s13353-011-0057-x

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Schon, K., & Tischkowitz, M. (2017). Clinical implications of germline mutations in breast cancer: TP53. Breast Cancer Research and Treatment, 167(2), 417–423. doi:10.1007/s10549-017-4531-y

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Suhail, Y., Cain, M. P., Vanaja, K., Kurywchak, P. A., Levchenko, A., Kalluri, R., & Kshitiz. (2019). Systems biology of cancer metastasis. Cell Systems, 9(2), 109–127. doi:10.1016/j.cels.2019.07.003

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Swale, V. J., & Quinn, A. G. (2000). Tumour suppressor genes. Clinical and Experimental Dermatology, 25(3), 231–235. doi:10.1046/j.1365-2230.2000.00620.x

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Zhu, Y., Wu, J., Zhang, C., Sun, S., Zhang, J., Liu, W., … Zhang, Z. (2016). BRCA mutations and survival in breast cancer: An updated systematic review and meta-analysis. Oncotarget, 7(43), 70113–70127. doi:10.18632/oncotarget.12158

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