10.6 Implications

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Cancer genome sequencing has had and will continue to have widespread impact on scientific research, the health care system and on the regular individuals who may one day be diagnosed with cancer.

 

Impact of the Health Care System

 

The introduction of cancer genome sequencing has changed the framework of the health care system (10). Next generation sequencing (NGS) has become an additional and extremely beneficial tool in every clinician’s tool box. Clinicians will now, not only send tumor and tissue sample to pathologist for a pathology reports, or lab technicians for screening for cancer markers, but also to genetics labs for sequencing (10). This additional tool will give clinicians a greater scope of information regarding what mutations are present in tumor samples, which will then help inform their diagnosis and treatment decisions (12).

 

Improved Methods of Screening and Diagnosing Disease

 

With the introduction of NGS techniques clinicians will be better able to screen and diagnose patients for with a higher resolution and adjustable specificity in a shorter amount of time than what current techniques, such as microarray, offer  (12). In the past, risk of developing cancer or a disease was assessed by investigating a patient’s family tree and determining whether any family members had or died of an ailment that may genetically predispose the patient and increase their risk of obtaining the same or other diseases (12). However, for patients with no family history this method was not helpful (12). In addition to obtaining a family history, the genomes of multiple patients can be sequenced at once for multiple mutations using NGS, and the presence of variants and single nucleotide polymorphisms can be compared to reference genomes to provide more comprehensive assessment of risk (12). In addition, NGS which are much more sensitive to rare variations provide a more accurate method of screening for bio-markers or the presence of mutations within tissue or blood that are associated to diseases (12). This is crucial as early disease detection has been instrumental in reducing mortality rates especially in cancer where 90% of deaths are cause by metastasis (12).

 

NGS also provide an alternate mechanism to grade diseases (13). In the past, the stage of cancer was graded by preforming a biopsy and either looking at the tissue under the microscope, karyotyping, or sequencing specific regions of the genome (13). Now, with NGS one can sequence the entire genome and see all the variations and mutations that been acquired (13). With this compressive picture of the tumor heterogeneity, the stage of the disease has progressed can be determined with more information. The grade of a tumor or disease may have profound impacts on how the patients is treated. The presence of certain mutations may alter the physician that more or less severe treatments are necessary and give patients a better chance of survival. This has led to the development of personalized medicine. 

 

 

Personalized Medicine

 

Personalized medicine is one of the goals for disease treatment. It has been known for some time that cancer is a very heterogeneous disease that is unique to the individual. No one treatment can or will cure everyone diagnosed with the same cancer. Thus, with the addition of cancer genome sequencing science has gotten one step closer to understanding each patient on a unique and highly specific level, and using that knowledge to determine the best treatment. Personalized medicine is discussed in detail in Chapter 9.

 

Cancer Genome and Application to Alternative Fields

      

Another important implication of cancer genome sequencing involves the subsequent translation of novel information to the general public, and the impact on health care decisions in the field of preventative medicine. Individuals with a history of familial or hereditary cancer in their families are slowly becoming more aware of options for assisted reproduction and the ability to select embryos without the cancer-predisposing mutations that run in their family through preimplantation genetic diagnosis (PGD) (6). PGD is performed on embryos, that result from in vitro fertilization (IVF), around 6 days post conception (6). After genetic testing the selected embryo is then transferred and implanted in the uterus (6). More specifically, the genetic diagnosis is made from a single cell and requires PCR amplification techniques (9). These single cell PCR techniques are sensitive to contamination and other difficulties, and are presently performed in specialized laboratories (9). As the understanding and knowledge of the cancer genome increases, it will increase the demand and scope for genetic counseling in general, and in the specific areas of prenatal diagnosis or PGD. This is combined with the expanding availability of new technologies and clinical genetic tests that predict cancer risk, as well as improved prospects for childbearing after cancer treatment (6).

 

PGD has been preformed for both cancer susceptibility syndromes, such as Li-Fraumeni syndrome, and for the detection of inherited mutations such as NF1 or RB1 (6). Interestingly enough, there is very little literature reporting the use of PGD for hereditary predisposition to breast and ovarian cancer, even though the BRCA1/2 mutations have been known for years (6). The relatively infrequent use of PGD is related to several important concerns. Some are related to age of onset for particular cancers; it may be deemed more applicable to life threatening childhood cancers with a significantly higher penetrance, while it’s use in cancers that occur later in life pose an ethical debate (6). Other ethical considerations involve the hypothesized further extension of PGD testing for sex selection or multifactorial disorders such as depression (6). These concerns are part of a much larger discussion involving the role of economic status, the extent of contact with specialists and the ability of oncologists to initiate discussions regarding assisted reproduction technology (6). Additionally, PGD is very costly and is only reimbursed by the health care systems in a few countries (7). For example, insurance companies in the USA may state that while they will cover IVF in the context of infertility, they don’t feel it is “medically necessary” to identify embryos through PGD and select them based on a potential predisposition to cancer (6). It should be noted that the long-term implications of assisted reproduction and IVF are still unknown, and there is concern regarding the possibility of imprinting defects due to epigenetic dysregulation (8). Due to the inherent link between PGD and IVF, PGD is also affected by the limitations of IVF. Lastly, there are concerns regarding the implications of hormone stimulation during IVF and it's effects on woman with a predispostition to hormone-sensitive cancers (7).     

 

The implications for the increasing knowledge of the cancer genome have widespread effects on the health care system, including the effect on genetic testing advances and assisted reproductive technology. More research needs to be conducted to assess the public awareness and views on PGD, the potential strategies for the application of PGD in the context of cancer, and strategies to deal with the increasing influx of cancer genome information through cancer genome sequencing and research.

 
 

Impact of Genome Sequencing on Scientific Knowledge and Discovery

 

 

Whole genome sequencing technology has revolutionized scientific discovery. With the introduction of NGS scientists have been able to identify a range of mutations that commonly occur in cancer tissue as well as other diseases (11). Using genome sequencing for a variety of tissue types and species, scientists have been able to discover new information and resolve questions about basic biological processes that have been unanswered for decades (11). In many diseases as well as cancer the underlying mutation or “driver” is often not completely clear, or remains unknown. Using NGS the underlying genetic component of diseases, and even behaviors, can be uncovered (11).

In cancer alone NGS has been used to further understanding of cancer initiation, progression, metastasis and treatment resistant (13). Using tissue and cells from patients at a variety of stages of cancer (primary, metastatic, treatment resistant) the life span or “evolution” of the cancer can be mapped (11). Not only does this reveal the mutation rate it can also further inform studies on disease mechanisms, and provide new possible therapeutic targets (11). As previously mentioned the sections above, cancer and many other maladies are heterogeneous and often inherently unique to the individual carrier. Using whole genome sequencing, rare disease can be studied comprehensively. Unlike many other molecular techniques, whole genome sequencing produces more data than can be computed by any one individual (12). While this may be a drawback of the technology it also speak volumes about the quantity of information that can be derived from the smallest of samples. 

 

Synthetic Lethality

 

Synthetic lethality occurs when the presence of one mutation still allows for cell viability, but when that mutation is present in combination with one or more other mutation(s) it leads to lethality. In a synthetic lethality genetic screen, a mutation that confers the desired phenotype must be tested against other mutations to rule out lethality. This approach when applied to cancer therapy seeks to identify and exploit these relationships to target tumor suppressor genes and reduce side effects. This would meet both goals of of personalized treatments based on cancer genomics: increased efficiency and reduced toxicity.By identifying and targeting a gene product that is synthetically lethal to a cancer-causing mutation, cancer cells would be targeted specifically and toxic side effects in normal cells minimized. The key to the development and effective use of this as a therapeutic option is the advancement of genetic technologies for exploring tumor mutations and gene function. For instance high throughput genome wide RNA interference screens can be used to identify potential targets (1). 

Synthetic lethality is more common for loss-of-function alleles, making tumor suppressors a likely target. For example, a mutation in a tumor suppressor may produce a lethal phenotype if and only if a mutation in a redundant gene product, same pathway or parallel pathway is introduced. Targeting a protein that is synthetically lethal to a cancerous mutation is a possible solution for the problem of targeting downregulated or ablated (and therefore “undruggable”) tumor suppressor gene products (1, 2). It is also selective such that only cells containing the tumor suppressor mutant gene would be affected.However, it is also possible to have synthetic lethality with a gain-of-function allele. For instance, if one gene is mutated and becomes overexpressed, a second gene product may become necessary for cell survival (1). Therefore, there is also potential for targeting oncogenes. Conversely, an earlier mutation may suppress the potential lethality of a later mutation (1). This has been coined “oncogene addiction”, in that the cancerous cell has become reliant on an earlier mutation to survive. If the original mutation were silenced, deleterious effects of subsequent mutations could be uncovered (1).

Synthetic lethality can be screened for using chemical pertubations or RNAi. The two cell lines differ only by mutation of a tumor suppressor. Chemicals or RNA's that produce a cytotoxic effect on the TSG-/-, but not WT cells, identify synthetic lethalities of potential therapeutic value. This filters compounds and genetic targets by selectivity. (1)

 

Of note is the fact that some interactions may be cell or tissue-specific or limited to a certain range of conditions. Therefore in vitro experimentation must guide extensive in vivo validation (1). It is difficult to identify targets that are translatable to a treatment setting, and where the effects of gene silencing in a screen are the same as silencing by drugs in a pharmacological setting (2). Other factors such as epigenetics, tumor heterogeneity and microenviornment may reduce the applicability of synthetic lethal targets (2).

Example:

Theoretical: Synthetic lethal interactions may be based on the particular cell cycle checkpoints. For instance, in cells with a p53 mutation, cells would be more susceptible to caffeine-based inhibition of ATR, a protein whose absence would encourage premature chromosomal condensation in S phase (1).

Efficacious in the lab: In an epigenome screen, BRM, which codes for an ATPase of the SWI/SNF chromatin remodelling complex, was found to be essential in tumor cells with BRG1 mutations. This selective dependency was also demonstrated in vivo. This is an example of selective paralog dependency as both BRM and BRG1 are catalytic subunits of the SWI/SNF complex (3). Growth suppression was observed in vitro and in vivo when BRG1-deficient tumor xenografts were treated subjected to BRM depletion via RNAi (4). BRG1-deficient tumor cells without other viable therapeutic targets are present in 15.5% of non small cell lung carcinomas (4).

Efficacious in patients: Tumors with BRCA1/2 mutations (tumor suppressors whose normal function is DNA repair) are sensitive to drugs targeting the PARP DNA-repair enzyme. Resistance to PARP-inhibitors can be acquired if BRCA1/2 function is regained (2). This shows that the mechanism of action was synthetic lethality.

As an explanation for drug effectiveness: Geldanamycin interferes with heat shock protein 90 (Hsp90), which is a chaperone protein that folds signaling proteins. Normally functioning Hsp90 buffers mutations by binding abnormal proteins. Therefore, by blocking Hsp90 many deleterious mutations in cancer cells may be uncovered and prove lethal for tumor cells. Normal cells do not exhibit the genetic instability critical to this process, making them less likely to suffer cytotoxic effects (5).

 

 

Whole genome sequencing has not only impacted understanding of biological diseases it has also greatly impacted scientific discovery in a much simpler way. The sequencing technique and methodology itself has rapidly progressed scientific knowledge. With the addition of NGS, knowledge which years ago would have taken decades to acquire is now only taking days to acquire and less than months to decipher and make sense of. Moreover, these discoveries are being made at a fraction of the cost. These new technologies can examine many samples at the same time and acquire data on a variety of information on the DNA sequence as well epigenetic modifications. Additionally, due to large output of data and new sequence databases which include sequences of thousands of genes and genomes targeted molecular biology has become more accurate. In the past when scientists were interested in studying a gene via PCR, knockdown or transgene insertions they would have to create primers and inserts based on homologous genes from well studies organisms. However, now, scientist can simply refer to databases which have sequence their gene of interest and create highly specific probes, primes or vectors for much more accurate targeting or amplification.

 

 

Repercussions

 

 

While the positive impacts of genome sequencing are unquestionable there remains some less favorable effects. Each sample, tissue, or patient tumor that is sequenced produces copious amounts of information. Depending on the depth and sensitivity of the sequencing technology the statistical significance of the some of the resulting data remain questionable (11). In addition, ethical questions have arisen to do with clinicians responsibility to reveal all genomic “abnormalities” or “variants of unknown significance that are revealed as currently the results may have no biological significance (11). Moreover, screens and whole genome sequences are revealing more information about individual’s genetic background and risks of developing disease. While these genome sequencing can identify individuals at high risk of developing cancer, they do not provide any guarantees (12). Thus, increased sensitivity and increased whole genome sequencing may place additional burden on the health care system and increase the number patients who choose to preform preventive surgeries or treatments (11). In addition, more ethical questions regarding privacy and the ramifications of results of genome sequencing has been raised. In some countries where health care requires patients to disclose any information that may impact their health before being approved of health coverage the consequences and regulations regarding disclosure of the genome sequence must be determined (12). Therefore, it is clear that the addition of this new technology will have broad ramifications not only within scientific research but also on an individual level. 

 

 

Conclusion

 

 

Genome sequencing technologies and the resulting whole genome sequence has profoundly impacted scientific discovery, disease treatment, and raised ethical questions about how the comprehensive results should be relayed to patients and raised question about privacy and confidentiality. NGS has greatly changed how scientific research is preformed and provided scientists and clinician an essential tool that has and will continue to be used to benefit patients suffering from disease and those who are at risk.

 

References

1) Kaelin WG. 2005. The concept of synthetic lethality in the context of anticancer therapy. Nature Reviews Cancer, 5(9): 689-98.

2) Nijman SM, Friend SH. 2013. Potential of the synthetic lethality principle. Science, 342(6160): 809-11.

3) Hoffman GR, Rahal R, Buxton F, Xiang K, McAllister G, Frias E, Bagdasarian L, Huber J, Lindeman A, Chen D, Romero R, Ramadan N, Phadke T, Haas K, Jaskelioff M, Wilson BG, Meyer MJ, Saenz-Vash V, Zhai H, Myer VE, Porter JA, Keen N, McLaughlin ME, Mickanin C, Roberts CW, Stegmeir F, Jagani Z. 2014. Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. Proceedings of the National Academy of Sciences, 111(8): 3128-33.

4) Oike T, Ogiwara H, Tominaga Y, Ito K, Ando O, Tsuta K, Mizukami T, Shimada Y, Isomura H, Komachi M, Furuta K, Watanabe SI, Nakano T, Yokota J, Kohno T. 2013 A synthetic lethality-based strategy to treat cancers harboring a genetic deficiency in the chromatin remodeling factor BRG1. Cancer Research, 73: 5508.

5) Garber K. 2002. Synthetic Lethality: Killing Cancer with Cancer. Journal of the National Cancer Institute, 94(22): 1666-8.

6) Offit, K., Kohut, K., Clagett, B., Wadsworth, E.A., Lafaro, K.J., Cummings, S., White, M., Sagi, M., Bernstein, D., Davis, J.G. Cancer genetic testing and assisted reproduction. Journal of clinical oncology. 2006. 24(29): 4775-4782.

7) Offit, K., Sagi, M., Hurley, K. Preimplantation genetic diagnosis for cancer syndromes. JAMA. 2006. 296(22): 2727-2730.

8) Arnaud, P., Feil, R. Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth defects Res C Embryo Today. 2005. 75:81-97.

9) Coskun, S., Alsmadi, O. Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosis. Prenatal diagnosis. 2007. 27: 297-302.10) Shyr, Derek, and Qi Liu. "Next Generation Sequencing in Cancer Research and Clinical Application." Biological procedures online 15.1 2013.

10)Shyr, Derek, and Qi Liu. 2013. Next generation sequencing in cancer research and clinical application. Biological Procedures Online 15 (1): 4-.

 11)Wu, Wei, Hani Choudhry, and SpringerLink ebooks - Biomedical and Life Sciences. 2013. Applications of next generation sequencing in cancer research: Vol. 1: Decoding the cancer genome. New York: Springer New York.

 12) Patel, Lalit R., Matti Nykter, Kexin Chen, and Wei Zhang. 2013. Cancer genome sequencing: Understanding malignancy as a disease of the genome, its conformation, and its evolution.Cancer Letters 340 (2): 152.

13) Xuan, Jiekun, Ying Yu, Tao Qing, Lei Guo, and Leming Shi. 2013. Next-generation sequencing in the clinic: Promises and challenges. Cancer Letters 340 (2): 284.