b'Identification of genetic mutations in very little tumor sample isused human glioma cell lines to develop a Sanger sequencing leading to some exciting revelations in cancer detection. Chalelaassay accompanied by RT-qPCR with a TaqMan Assay to test et al. used multiple genetic technologies to demonstrate thatfor promoter mutations in a telomerase reverse transcriptase driver mutations can appear in histologically noncancerouspromoter gene (TERTp) that is associated with the diagnosis and cells, even without producing clonal expansion and withoutpoor prognosis in gliomas. They used the assay to analyze the coincident mutation in the primary tumor cells. They used Sangerfrequency of TERTp mutations among different types of gliomas, sequencing to identify specific exons within the EGFR or KRAStissue preparations, patient ages, tumor locations, and pathologic oncogenes. castPCR technology and, independently, qPCRstages. The authors suggested that a fast, straightforward, and confirmed mutation status, and dPCR confirmed the presenceaccurate method such as Sanger sequencing may aid in earlier of the EGFR or KRAS mutations. Using these technologies, DNAdiagnosis and prognostic assessment of gliomas [7]. extracted from normal lung tissue and lung adenocarcinomaAcute myeloid leukemia (AML) exemplifies the need for rapid (ADC) revealed EGFR or KRAS mutations in nontumoral lung cellsand sensitive cancer mutation detection. Approximately 50% even in patients with early-stage lung adenocarcinoma who hadof AML patients present with normal karyotypes; however, tested negative for those mutations [5]. multiple cellular regulatory genes carry mutations that impact Liquid biopsy prognosis and survival. Olarte et al. used castPCR technology Solid-tissue biopsy is the most widely used method for detectingto detect mutations in the isocitrate dehydrogenase (IDH) genes and categorizing tumors. However, tissue biopsy requiresfrom AML patient samples. castPCR technology revealed a invasive tissue extraction, which is not always feasible, and thehigher frequency of IDH1 mutations than IDH2 mutations; IDH1 information it yields is limited, both spatially and temporally. Solid- mutations are associated with unfavorable prognosis, while IDH2 tumor biopsies may not capture complex tumor heterogeneitymutations are not associated with any change in survival. Some or changes in malignancy over time. Noninvasive access toIDH mutations present opportunities as therapeutic targets [8]. evidence of cancer mutations via circulating tumor DNA (ctDNA)Quicker detection of important mutations would enable patients in bodily fluids such as blood, lymph, urine, and semen can be anto benefit from targeted therapies sooner.important tool to detect cancer at early stages. Liquid biopsy canAs understanding the genetic factors that impact disease also be a valuable approach to track efficacy of therapy over theamong different geographic and ethnic populations becomes course of treatment, monitor patients for relapse or metastasis,more important, so does understanding population structures or assess malignancy in inaccessible tissues. Detecting ctDNA isto develop new approaches for individualized cancer detection. often challenging because the DNA molecules bearing the cancerMighri et al. conducted haplotype analysis using NGS and mutations are only a small fraction of the total circulating cell-freeSanger sequencing to investigate the genetics and origin of DNA (cfDNA) collected in the liquid sample.the BRCA1-c.211dupA mutation, which has been found only in The high sensitivity of Absolute Q dPCR assays with microfluidicTunisian families with breast cancer. The mutation has a severe dPCR is exceptionally well suited to detecting mutations in cfDNAphenotype, including onset at a young age, underscoring in liquid biopsies. In a recent study, Siggillino et al. assessedthe need for early detection. Breast cancer patients with the ongoing response of non-small cell lung cancer (NSCLC)BRCA1-c.211dupA mutation are already using their diagnosis to to targeted treatment by measuring plasma levels of EGFRguide therapeutic decisions [9].mutations. They conducted quantitative analyses comparingNew approaches in data analysis may also offer improvements dPCR with TaqMan Assays, amplification-refractory mutationin genetic tests for cancer detection. In some cases, imaging system PCR, and peptide nucleic acid (PNA)-mediated PCRmethodologies used to diagnose solid tumors can result in false clamping. In their studies quantifying EGFR mutation detection,positives due to benign tissue masses. De Rienzo et al. sought to dPCR with TaqMan Assays yielded the highest sensitivitydetermine whether molecular testing could reduce false positives. and best correlation of liquid biopsy with tissue biopsy. TheThey developed a test based on gene expression ratio using authors concluded that dPCR is a robust method for absoluteApplied BiosystemsSYBR Green dyebased RT-qPCR to quantitation and monitoring of the evolution of mutationsdetermine expression levels of several genes associated with lung over time [6].cancer in normal and cancerous lung tissues. They determined the test to be a very sensitive and specific approach to reliably Genetic tests differentiate normal lung from cancerous tissue [10]. Sanger sequencing is widely used to interrogate genes for small fragment mutations. It can be a fast, accurate, and sensitive approach to detecting cancer mutations. Bai et al. Contents 6'