ran dan sequencingNext generations sequencing (NGS) has expanded beyond research applications to deliver clinically actionable information that can effectively guide therapeutic decision-making. Cancer researchers today seek to translate the immense volume of genomic data to clinical applications [1]. Within a clinical perspective, NGS has the potential to fill major gaps in molecular markers for the diagnosis, prognosis, and management of many human cancers. By creating a profile of a patient's specific tumor mutations, doctors can make informed decisions about the type of therapy a patient should receive. Since NGS is effective at targeting specific genes or pathways and correcting for particular mutations, characterizing a patient's cancer may soon be a required regiment in a patient's cancer therapy. Having this DNA sequence data available at treatment centers could provide clinicians with a complete view of the genetic alterations at the DNA and RNA levels. This can help identify specific characteristics in each patient, and discover new actionable biomarkers, effectively paving the road toward personalized medicine.

Non-invasive diagnostic tools for cancer using circulating DNA

Recently, sequencing of circulating cell-free tumor DNA (ctDNA) has emerged as a promising and noninvasive tool for quantifying tumor burdens and monitoring treatment responses. ctDNA is present in healthy subjects at an average concentration of 30 ng/ml of blood (ranges 0-100) [2], representing an average of 5,000 (ranges 0-15,000) genome equivalents per milliliter of blood. Two main biological mechanisms have been proposed for the release of ctDNA in the blood: 1.) apoptosis/necrosis or 2.) release of intact cells in the bloodstream [3].

ctDNA carries information regarding tumor mutations and tumor burden; thus, it has potentially transformative applications in cancer management.

High levels of ctDNA in cancer patients are a bad prognostic indicator. Changes in ctDNA levels may indicate response to therapy or disease progression, and indeed may prove to be the earliest indicator of changes to tumour burden [4] (see Figure 1).

Figure 1. Clinical procedure for measuring ctDNA as a personalized biomarker.

measuring ctDNA as a personalized biomarker

 

Image courtesy of: Cancer Research UK Cambridge Institute.<http://www.cruk.cam.ac.uk/>

Studies since 1998 have shown the feasibility of using ctDNA as a tool for cancer diagnosis and prognosis. Using traditional technologies, different types of DNA alterations including point mutations, DNA methylation, microsatellite instabilities, and chromosomal alterations have been reported in ctDNA of a large variety of cancer types including colorectal, pancreas, lung, bladder, head and neck, and liver [5-10]. However, most of these studies targeted a single gene or a small subset of genes with known alterations.

With the power of NGS technologies, researchers are able to identify genome-wide de novo tumor-derived alterations in ctDNA. Last year, a study performed whole-genome sequencing of ctDNA samples collected from colon and breast cancer patients. Their results demonstrated that whole-genome analysis of ctDNA could be used as a tool for detecting structural alterations specific to patients [11]. By sequencing a single gene using NGS, researchers demonstrated an ability to detect low-frequency mutations (as low as 2%) in ctDNA, with great sensitivity of measuring ctDNA, cancer antigen 15-3, and circulating tumor cells to monitor tumor burden in patients with metastatic breast cancer using whole-genome or targeted sequencing [12]. Their data showed that the sensitivity of ctDNA was superior to that of other circulating biomarkers and that ctDNA had a greater dynamic range that correlated with changes in tumor burden.

In more than half the patients, ctDNA provided the earliest measure of treatment response:  Recently Murtaza and colleagues showed that whole exome sequencing of ctDNA from patients with advanced breast, ovarian, and lung cancers [13]. Their data confirmed the genome-wide representation of the tumor genome in plasma and identified increased representation of mutant alleles associated with the emergence of therapy resistance, including mutations inPIK3CA and RB1.

Sequencing ctDNA has tremendous potential to non-invasively detect cancer. This approach represents a kind of liquid biopsy alternative, which could eventually replace invasive biopsies as a means to track the evolution of a patient's cancer. Also, ctDNA measurements may serve as important biomarkers for real-time monitoring of the efficacy of treatment plans in cancer patients. This could accelerate drug development as well as to identify subpopulations of patients who could benefit from alternative therapies.

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Therapeutic Opportunities

clinical genome sequencing

There is little doubt that NGS technologies have enabled the discovery of new varieties of biomarkers, including known biomarkers discovered in new cancer types [14]. The number of drugs in oncology with either FDA approval or under development is still limited, but is increasing over the last several years [15].  The question for researchers now is, how do we translate the encylopedia of sequencing data into actionable and effective therapies?

Potential applications for putative cancer biomarkers would allow for predictive, prognostic, and pharmacogenomic biomarkers that will provide decision-making support for answering questions about who should be given treatment and which therapy should be chosen.

Studies on the therapeutic applications of inhibitors targeting KIT mutations provide one such illustrative example. Oncogenic KIT mutations are common in testicular cancer, gastrointestinal stromal tumors (GISTs), and melanomas [16-19]. In particular, these KIT mutations lead to ligand-independent kinase activation [20]. As a result, they are becoming a crucial diagnostic biomarker for such tumors. Already so far, the US Food and Drug Administration (http://www.fda.gov/drugs/default.htm) has approved eight tyrosine kinase inhibitors (TKIs) targeting KIT, and many more are in clinical trials. Several have already been approved or tested in GIST and melanoma patients.

The KIT story provides an important model of how detailed mutation maps can be applied to the decision making process for effective cancer care.

More recently, researchers have proposed new treatment options that combine targeted therapy and immunotherapy in cancer treatment [21]. Targeted approaches inhibit essential molecular pathways that are required for tumor growth and survival; by contrast, immunotherapy aims to stimulate a host immune response that effectuates long-lived tumor destruction. Analyzing the strengths and weaknesses of targeted therapy and immunotherapy suggests that the two approaches may have complimentary roles in cancer treatment and that combined therapy could prove synergistic and lead to more effective treatments [22].

 

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[5] Esteller M, Sanchez-Cespedes M, Rosell R, et al. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Research. 1999. 59:67-70.

[6] Kirk GD, Lesi OA, Mendy M, et al. 249ser TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma. Oncogene. 2005. 24:5858-67.

[7] Mulcahy HE, Lyautey J, Lederrey C, et al. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clinical Cancer Researcher. 4:271-75.

[8] Salbe C, Trevisiol C, Ferruzzi E, et al. Molecular detection of codon 12 K-ras mutations in circulating DNA from serum of colorectal cancer patients. Int. J. Biol. Markers. 2000. 15:300-7.

[9] Utting M, Werner W, Dahse R, et al. Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: a minimally invasive method for the detection of bladder cancer. Clinical Cancer Researcher. 8:35-40.

[10] Leary RJ, Sausen M, Kinde I, et al. Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. 2012. Science of Translational Medicine. 4:162ra154.

[11] Forshew T, Murtaza M, Parkinson C, et al. noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Science of Translational Medicine. 2013. 4:136ra68.

[12,13] Murtaza M, Dawson SJ, Tsui DW, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 497:108-12.

[15] Wheeler DA, Wang L. From human genome to cancer genome: the first decade. Genome Res. 2013. 23:1054-62.

[16] Kemmer K, Corless CL, Fletcher JA, et al. KIT mutations are common in testicular seminomas. 2004. American Journal of Pathology. 164:305-13.

[17] Coffey J Linger R, Pugh J, et al. Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: report of 220 tumors and review of literature. Genes Chromosome. Cancer. 2011. 47:34-42.

[18] Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumors: origin and molecular oncology. Nature Reviews Cancer. 2011. 11:865-78.

[19] Garrido MC, Bastian BC. KIT as a therapeutic target in melanoma. Journal Investigational Dermatology. 2010. 130:20-27.

[20] Gajiwala KS, Wu JC, Christensen J, et al. KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients. National Academy of Sciences. 2009. USA. 106:1542-47.

[21,22] Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nature Reviews Cancer. 2012. 12:237-51.