2.2.2　Tumor Diagnosis

Biopsy: A piece of tissue is removed to be examined under a microscope to determine if it is cancerous.

CT scan and MRI scan: These are done to ascertain the exact location of the tumor in the body.

Bone marrow biopsy: This is done if there is suspicion of leukemia or lymphoma.

Tumor marker tests: These are biomarkers which can be found at elevated levels in blood and urine and even body tissue of the patient.

CBC (complete blood count): This is done to measure types of cells and their abnormalities.(https://www.daowen.com)

The development and continuous evolution of NGS techniques have revolutionized genomic as well as transcriptomic analysis.NGS platforms offer deep sequencing, which is capable of detecting very low frequency genetic variants, as well as massively parallel sequencing that can rapidly and comprehensively cover the human genome.Various NGS methods are utilized for whole-genome sequencing, whole-exome sequencing, targeted sequencing and RNA-seq.In turn,these applications are used to identify mutations in coding and non-coding genomic regions as well as impaired transcriptome dynamics.These features have propelled NGS to the forefront of cancer biomarker discovery where high sensitivity and massively parallel sequencing allow rapid detection of somatic and germline mutations [4].

NGS technologies are routinely utilized in clinical research studies, such as The Cancer Genome Atlas project, with the goal of discovering variant patterns that can serve as cancer biomarkers.Consequently, the implementation of NGS in translational research has led to the generation of large, cancer-specific genomic data sets and the identification of genetic variants, including very low frequency mutations, which have diagnostic, prognostic and therapeutic utility.Although cancer onset can be associated with key driver mutations, disease etiology and progression depend on the patient-specific combination of genetic variants.This compound effect of somatic and germline mutations is addressed through Precision Medicine initiatives that utilize genome-wide or targeted NGS sequencing to establish the genomic profile of individual patients.For example, NGS testing performed in NSCLC patients can detect the low frequency EGFR T490M mutant, which is resistant to gefitinib and erlotinib therapy, and can guide medical decisions [4, 15].

Clonal evolution and continuous tumor remodeling lead to tumor heterogeneity, which presents a tremendous challenge in oncology with respect to understanding disease etiology, progression and treatment options.Although conventional biopsy specimens are not representative of complete tumor heterogeneity, they often consist of normal cells and multiple clonal cell types that harbor distinct genomic mutations.Distinguishing low frequency mutations from background signal is difficult in these complex samples.However, NGS techniques can successfully detect clonal variants through deep sequencing and identify impaired signaling pathways and potential therapeutic targets.A more comprehensive assessment of tumor heterogeneity can be achieved through liquid biopsy specimens, where NGS methods are used to sequence low abundance circulating tumor DNA (ctDNA) that originates from clonal cells throughout the tumor and is representative of primary and metastatic sites.Additionally, liquid biopsy testing is less invasive than conventional biopsies and can be performed routinely to monitor disease progression and response to therapy, rendering it the primary focus of cancer biomarker translational research [4, 16].

Implementation of high sensitivity and ultra-high throughput NGS techniques has generated tremendous amounts of genomic data across cancer types and led to the identification of a myriad of genetic variants that can serve as cancer biomarkers.However, the impact that genetic mutations have on the transcriptome and proteome of cells cannot be inferred directly from sequencing analysis, especially when genomic variables are localized to non-coding regions.Additional challenges with genomic cancer biomarkers stem from lack of standardization pertaining to NGS techniques and difficulties with the validation of rare genetic cancer biomarkers where sufficiently large patient cohorts cannot be obtained.Another limitation of NGS is the cost of performing whole genome or exome sequencing that is necessary for cancer biomarker discovery.However, despite these challenges the US FDA in 2016 approved the first NGS clinical test, Clono SEQ, to be used for detection of minimal residual disease in patients with acute lymphoblastic leukemia and multiple myeloma.In summary, genomic analysis has led to the discovery of many genetic variants that are currently used as cancer biomarkers in lab-developed tests, but in the absence of corresponding proteomic and cell signaling data, these mutations have limited diagnostic,prognostic and therapeutic value [4].