Along with the dramatic development of molecular diagnostic testing for the detection of oncogene variations, reference materials (RMs) have become increasingly important in performance evaluation of genetic testing.
OBJECTIVE:
In this study, we built a set of RMs for genetic testing based on next-generation sequencing (NGS).
METHOD:
Solid tumor tissues were selected as the samples of RMs for preparation. NGS was used to determine and validate the variants and the mutation frequency in DNA samples. Digital PCR was used to determine the copy numbers of RNA samples. The performance of the RMs was validated by six laboratories.
RESULTS:
Thirty common genetic alterations were designed based on these RMs. RMs consisted of a positive reference, a limit of detection reference, and a negative reference. The validation results confirmed the performance of the RMs.
CONCLUSION:
These RMs may be an attractive tool for the development, validation, and quality monitoring of molecular genetic testing.
KhodakovDWangCZhangDY. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches. Advanced Drug Delivery Reviews. 2016; 105: 3-19.
2.
MerrickBA. Next-generation sequencing data for use in risk assessment. Current Opinion in Toxicology. 2019; 18: 18-26.
3.
BentleyDRBalasubramanianSSwerdlowHP, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature.2008; 456: 53-59.
4.
RothbergJMHinzWRearickTM, et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature.2011; 475: 348-352.
5.
PinheiroLBColemanVAHindsonCM, et al. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification. Anal Chem.2012; 84: 1003-1011.
6.
DequekerERamsdenSGrodyWW, et al. Quality control in molecular genetic testing. Nature Reviews Genetics.2001; 2: 717-723.
7.
LanY-TJen-KouLLinC-H, et al. Mutations in the RAS and PI3K pathways are associated with metastatic location in colorectal cancers: Mutations of EGFR Pathways in CRCs. J Surg Oncol.2015; 111: 905-910.
8.
DimriMSatyanarayanaA. Molecular signaling pathways and therapeutic targets in hepatocellular carcinoma. Cancers.2020; 12: 491.
9.
LiYXiongH. Correlation of LAGE3 with unfavorable prognosis and promoting tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways. BMC Cancer.2022; 22: 298.
10.
MacConaillLECampbellCDKehoeSM, et al. Profiling critical cancer gene mutations in clinical tumor samples. PLoS One.2009; 4: e7887.
11.
LynchTJBellDWSordellaR, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. New England Journal of Medicine.2004; 350: 2129-2139.
12.
CostaDBKobayashiSS. Whacking a mole-cule: Clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M. Transl Lung Cancer Res.2015; 4: 809-815.
13.
KobayashiYTogashiYYatabeY, et al. EGFR exon 18 mutations in lung cancer: Molecular predictors of augmented sensitivity to afatinib or neratinib as compared with first- or third-generation TKIs. Clin Cancer Res.2015; 21: 5305-5313.
14.
KrawczykPMlakRPowrózekT, et al. Mechanisms of resistance to reversible inhibitors of EGFR tyrosine kinase in non-small cell lung cancer. Contemp Oncol (Pozn).2012; 16: 401-406.
15.
WilsonPMLaBonteMJLenzH-J. Molecular markers in the treatment of metastatic colorectal cancer. The Cancer Journal.2010; 16: 262.
16.
RoockWClaesBBernasconiD, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: A retrospective consortium analysis. The Lancet Oncology.2010; 11: 753-762.
17.
Laurent-PuigPCayreAManceauG, et al. Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. JCO.2009; 27: 5924-5930.
18.
EngelmanJAZejnullahuKMitsudomiT, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science.2007; 316: 1039-1043.
19.
BardelliACorsoSBertottiA, et al. Amplification of the MET Receptor Drives Resistance to Anti-EGFR Therapies in Colorectal Cancer. Cancer Discovery.2013; 3: 658-673.
20.
JuLHanMZhaoC, et al. EGFR, KRAS and ROS1 variants coexist in a lung adenocarcinoma patient. Lung Cancer.2016; 95: 94-97.
21.
SharmaGMotaIMologniL, et al. Tumor resistance against ALK targeted therapy-where it comes from and where it goes. Cancers.2018; 10: 62.
22.
RielyGJKrisMGRosenbaumD, et al. Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clinical Cancer Research.2008; 14: 5731-5734.
23.
PaezJGJännePALeeJC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science.2004; 304: 1497-1500.
24.
TanC-SKumarakulasingheNBHuangY-Q, et al. Third generation EGFR TKIs: Current data and future directions. Molecular Cancer.2018; 17: 29.
25.
KeEEZhouQZhangQY, et al. A higher proportion of the EGFR T790M mutation may contribute to the better survival of patients with exon 19 deletions compared with those with L858R. J Thorac Oncol.2017; 12: 1368-1375.
26.
DingLGetzGWheelerDA, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature.2008; 455: 1069-1075.
27.
PaikPKArcilaMEFaraM, et al. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol.2011; 29: 2046-2051.
28.
PratilasCAHanrahanAJHalilovicE, et al. Genetic predictors of MEK-dependence in non-small cell lung cancer. Cancer Res.2008; 68: 9375-9383.
29.
ZhengDWangRPanY, et al. Prevalence and clinicopathological characteristics of BRAF mutations in chinese patients with lung adenocarcinoma. Ann Surg Oncol.2015; 22: 1284-1291.
30.
SchefflerMBosMGardiziM, et al. PIK3CA mutations in non-small cell lung cancer (NSCLC): Genetic heterogeneity, prognostic impact and incidence of prior malignancies. Oncotarget.2014; 6: 1315-1326.
31.
SamuelsYWangZBardelliA, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science.2004; 304: 554-554.
32.
FanJXiaZZhangX, et al. The efficacy and safety of alectinib in the treatment of ALK+ NSCLC: A systematic review and meta-analysis. OncoTargets and Therapy.2018; 11: 1105-1115.
33.
WangJMirzapoiazovaTCarol TanY-H, et al. Inhibiting crosstalk between MET signaling and mitochondrial dynamics and morphology: A novel therapeutic approach for lung cancer and mesothelioma. Cancer Biology & Therapy.2018; 19: 1023-1032.
34.
ZhangQZhangX-CYangJ-J, et al. EGFR L792H and G796R: Two novel mutations mediating resistance to the third-generation EGFR tyrosine kinase inhibitor osimertinib. Journal of Thoracic Oncology.2018; 13: 1415-1421.
35.
MorgilloFDella CorteCMFasanoM, et al. Mechanisms of resistance to EGFR-targeted drugs: lung cancer. ESMO Open.2016; 1: e000060.
36.
DoHDobrovicA. Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase. Oncotarget.2012; 3: 546-558.
37.
DoHWongSQLiJ, et al. Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates. Clinical Chemistry.2013; 59: 1376-1383.
38.
MyersMBMcKimKLMengF, et al. Low-frequency KRAS mutations are prevalent in lung adenocarcinomas. Per Med.2015; 12: 83-98.
