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Role of Next-Generation Sequencing in Cancer Diagnostics

The significant global burden of cancer has led to increased efforts to determine prevention strategies, early detection, and successful management of patients diagnosed with cancer. The latest data provided by the World Health Organization reveal cancer has accounted for 8.2 million deaths as of 2012¹. Early detection and successful treatment are considered important factors in reducing cancer incidence and mortality. Accurate and early diagnosis is necessary for an appropriate treatment that can be tailored to the individual patient.

Next-Generation Sequencing Overview

Sensitive, specific, high-throughput, and inexpensive diagnostic technologies bolster efforts to achieve early, accurate, and cost-effective cancer detection. The new technologies comprising next-generation diagnostics are keys to satisfying these requirements. A genetic technology that has become the gold-standard in cancer diagnostics is next-generation Sequencing (NGS). Next-generation sequencing differs from Sanger sequencing in that it is much faster (what took years with Sanger sequencing takes only weeks), requires less DNA samples, and is more cost-effective. With NGS, millions of samples can be simultaneously sequenced and analyzed.

Accurate Detection & Treatment

Given the genetic characteristic of cancer etiology, NGS is a key player in cancer diagnostics. Applying genetic testing for cancer has greatly improved the level of understanding of the multifaceted disease. Gene mutations associated with tumor formation and progression can be reliably detected using NGS. Ellison et al.²  have demonstrated accurate detection of BRCA1 and BRCA2 in fixed tumor tissue (validated by Sanger sequencing). The NGS strategy is conducive to the analysis of mutations with starting samples containing highly fragmented and lower levels of DNA.

Clinical tumor specimens are typically formalin-fixed and paraffin-embedded. The limitations that fixed-samples provide for genetic testing are overcome with NGS because this strategy involves the amplification of relatively short sequences. Also, the lower DNA levels obtained from fixed-tissue samples do not present major barriers for NGS. The technology may represent a revolution in cancer biology and medicine by providing information for the design of specific targeted drugs and a better prediction of clinical outcomes. Rare somatic mutations can also be better identified and distinguished from germline mutations.

NGS Sensitivity

A study by D´Argenio et al.³ also demonstrates the utility and sensitivity of NGS in the detection of mutations in BRCA1/2.  Next-generation sequencing analysis was compared with Sanger sequencing. Women were screened by both methods followed by bioinformatic analysis of the data. The results indicate that NGS is more sensitive in detecting BRCA1/2 variants than the Sanger method and may be considered very reliable for BRCA1/2 gene screening.

Targeted Diagnoses

Devarajan et al.4 used a targeted NGS approach to retinoblastoma diagnosis. Patient DNA samples (from blood or tumors) were sequenced. A bioinformatics approach was developed to detect pathogenic variants. The research team also wanted to distinguish between somatic and germline mutations. Detection of pathogenic variants was successful in 85% of the patients in the study. Distinguishing between somatic and germline mutations was also achieved. This demonstrated the clear potential of NGS for genetic/molecular diagnosis of retinoblastoma.


As technology advances, NGS has the potential to make the future of genome sequencing an integral aspect of personalized medicine. The sensitivity and specificity of genetically characterizing tumor cells in patients has profound implications regarding treatment choices and predicting potential responses. For example, in a study by Gleeson et al., 20155, the KRAS, NRAS, or BRAF mutations identified in the lymph nodes of a sample of patients with rectal cancer suggest that 42% of the patients would likely fail to respond to anti-epidermal growth factor receptor therapy. If treatment can be designed to target specific tumor genetic profiles, failed responses to therapies and poorer prognoses can be avoided.

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1   World Cancer Report 2014  (http://www.who.int/mediacentre/factsheets/fs297/en/)

2  Ellison G, Huang S, Carr H, Wallace A, Ahdesmaki M, Bhaskar S, Mills J. A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing. BMC Clin Pathol. 2015;24:15:5.

3  D’Argenio V, Esposito MV, Telese A, Precone V, Starnone F, Nunziato M, Cantiello P, Iorio M, Evangelista E, D’Aiuto M, Calabrese A, Frisso G, D’Aiuto G, Salvatore F. The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches. Clin Chim Acta. 2015;446:221-225.

4  Devarajan B, Prakash L, Kannan TR, Abraham AA, Kim U, Muthukkaruppan V, Vanniarajan A. Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma. BMC Cancer. 2015;15:320.

5  Gleeson FC, Kipp BR, Voss JS, Campion MB, Minot DM, Tu ZJ, Klee EW, Sciallis AP, Graham RP, Lazaridis KN, Henry MR, Levy MJ. Endoscopic ultrasound fine-needle aspiration cytology mutation profiling using targeted next-generation sequencing: personalized care for rectal cancer. Am J Clin Pathol. 2015;143:879-88.