Targeting drugs such as cetuximab and panitumumab have been widely used in clinic as effective therapeutic drugs for colorectal cancer. Clinical data show that patients with KRAS mutations have no significant effect on this monoclonal antibody drug, and only wild-type patients can benefit from it. Therefore, the KRAS gene mutation status is clinically regarded as an important therapeutic marker, which has a strong correlation with the prognosis and treatment effect of colorectal cancer. The 2009 National Cancer Comprehensive Network (NCCN) Colorectal Cancer Clinical Practice Guidelines stipulates that all patients with metastatic colorectal cancer must detect KRAS gene mutation status, and only KRAS wild type is recommended to receive EGFR targeted therapy. In the same year, the American Society of Clinical Oncology (ASCO) also issued the same clinical treatment recommendations as a molecular marker for tumor targeted therapy, which shows its important guiding significance. At present, KRAS genetic testing has been widely carried out clinically. We mainly evaluate the domestic KRAS gene mutation detection methods for reference in clinical selection.
1. Izinga elihle lokushintshwa kofuzo ku-KRAS kumdlavuza obala ngokwezibalo
Emdlavuzweni ongaguquki, isilinganiso sokuguquka kwesakhi sofuzo se-KRAS sifike ku-35% kuye ku-45%, kanti indawo enobungozi obukhulu ingu-codon 12 no-13 ku-exon 2, futhi kusekhona ezingandile ezifana no-61 no-146. indawo. Kunezindlela eziningi zokubona zokuguqulwa kwezakhi zofuzo ze-KRAS, kufaka phakathi ukulandelana okuqondile, ukuhlaziywa okuphezulu kokuncibilika kwejika (HRM), i-pyrosequencing, i-PCR yokulinganisa, i-mutation amplification block system (amplinc atio) nrefractorymutation system (ARMS), i-limited fragment length polymorphism (RFLP), i-polymerase chain reaction-single-strand conformation polymorphism analysis (i-PCR-singlestrand confomation polymorphism (PCR-SSCP), i-co-amplification endaweni ephansi ye-denaturation temperatur PCR (COLD-PCR) kanye nokuhlaziywa okuphezulu kokusebenza kwe-liquid chromatography, njll.
2. Ukuhlolwa kwezindlela zokuthola ukuguqulwa kwezinguquko ze-KRAS
1. Indlela yokulandelana okuqondile: Kuyindlela yakudala kakhulu yokuthola ukuguqulwa kwezakhi zofuzo ze-KRAS, futhi futhi kuyindinganiso yegolide yokuthola ukuguquka kwezakhi zofuzo. Indlela yokulandelana eqondile ngokususelwa kumgomo wokulandelana kwe-dideoxy ingabonisa intuitively kakhulu ushintsho lokulandelana kwezakhi zofuzo ngendlela yemephu eyisisekelo. Uhlobo lokuthola lubanzi kakhulu, futhi futhi kuyindlela yokuqala yokutholwa kokuguqulwa kokushintshwa. Ngaphandle kokuvela kwamapulatifomu wokulandelana kwesizukulwane esisha, izifundiswa ekhaya nakwamanye amazwe zisayisebenzisa imiphumela yokulandelana ngqo njengesikali ukukala nokunquma ukuthembeka kwendlela entsha. UGao Jing et al. Kusetshenziswe ukulandelana okuqondile ekutholeni ukuguqulwa kofuzo kwe-KRAS ne-BRAF ezigulini ezingama-966 ezinomdlavuza obala. Lokhu futhi kuwukuhlaziywa kokuguqulwa kwezakhi zofuzo kwe-KRAS ngesampula enkulu yasekhaya ebikiwe ezincwadini. U-Ling Yun nabanye bakholelwa ukuthi indlela yokulandelana eqondile iyindlela yokubona eqonde ngqo nephumelelayo yokuqonda isimo sokuguquka kwesakhi ngasinye, esingacacisa uhlobo lokuguquka, ikakhulukazi ekutholeni izinguquko ezingaziwa. Yize ukuzwela kwale ndlela kuncane kakhulu, kungathuthukiswa ngezindlela ezinjenge-microdissection ukukhulisa amaseli wesimila. Indlela yokulandelana ngqo nayo isetshenzisiwe ekutholeni ama-KRAS osayizi abakhulu besampula kwamanye amaqembu ocwaningo lwasekhaya. Kodwa-ke, ukuzwela okuphansi kuyinkinga enkulu yokulandelana ngqo. Uma kubhekwa imiphumela ebikwe eChina, izinga lokutholwa kokuguquka kwezakhi ngokulandelana okuqondile aliphansi. U-Liu Xiaojing et al. Uma kuqhathaniswa ukulandelana okuqondile kanye ne-peptide nucleic acid clamp PCR (PNA-PCR) futhi kutholakale ukuthi amacala angama-43 wokuguqulwa kwezakhi zofuzo kwe-KRAS atholwe ngokulandelana okuqondile. Ngaphezu kwalokhu kuguquka kwezakhi, iPNA-PCR nayo itholwe ngokulandelana okuqondile. Izinguquko eziyishumi zitholakale ohlotsheni lwasendle, futhi kwenziwa iziphakamiso zokunquma iziguli zohlobo lwasendle yi-PCR kanye nendlela eqondile yokulandelana ukuthola iziguli eziguqukayo. UQiu Tian et al. Kutholwe izibonelo zomdlavuza we-131 colorectal nge-fluorescent PCR-optimised oligonucleotide probe indlela kanye nendlela eqondile yokulandelana, futhi amanani amahle wokushintshwa kofuzo lwe-KRAS abengu-41.2% (54/131) kanye no-40.5% (53/131)). UBai Dongyu uphinde wakhuluma ngokuzwela kokuthola kwezindlela ezahlukahlukene. Ezigulini ezinomdlavuza ezibalelwa ku-200, ezingama-63 zitholwe yizinguquko ze-RT-qPCR, kanti izinga lokutholwa kokuguqula izinguquko kube ngu-31.5%; Amasampula ayi-169 alandelwa ngempumelelo ngokulandelana okuqondile kwamacala angama-50 okuguquka kwezakhi zofuzo, izinga lokutholwa kokuguquka kwezinguquko lalingu-29.6%. Yize indlela elandelanayo eqondile ingathola ngokunembile, ngokuqonde ngqo nangokuqondile isimo se-KRAS sokuguquka kwezakhi zofuzo, ukushiyeka kwayo okufana nezidingo eziphezulu zobuchwepheshe, izinqubo zokusebenza eziyinkimbinkimbi, kulula ukudala ukungcoliswa okuphambene, nokuchithwa kwesikhathi nokuchitha kanzima kwemiphumela nakho kusobala. Imvamisa ayikho imishini yokulandelana, futhi isampuli idinga ukuthunyelwa enkampanini ehambisanayo ukuze ihlolwe, okuthatha isikhathi eside futhi kubiza kakhulu, ngakho-ke kunemikhawulo emikhulu.
Indlela ye-Pyrosequencing:
Indlela ye-Pyrosequencing futhi iyindlela elula kakhulu yokuthola ukutholwa koguquko lwe-KRAS ngokuya ngokulandelana kokuzwela, izindleko zokuthola nesikhathi sokubika. Ukuphindeka kwale ndlela kungcono. Ngokwebalazwe eliphakeme elitholakele Ucwaningo lobungako bokuvama kokuguquka kwesiza esithile nokuqhathanisa phakathi kokuvama kokuguquka kwezindawo ezahlukahlukene kucacile shazi. Eminyakeni yakamuva, u-Ogino et al., UHutchins et al. Sisebenzise ubuchwepheshe be-pyrosequencing ukuhlola ukuguqulwa kwe-KRAS ezigulini ezinamasampula amakhulu omdlavuza obala. Imiphumela ikhombisa ukuthi ubuchwepheshe be-pyrosequencing buyithuluzi elinamandla lokuhlola iziguli ukuthola ukwelashwa okuhlosiwe. Ukuxilongwa kwe-tumor yamangqamuzana kunamathuba amaningi wokusebenza Izazi zasekhaya zisebenzise ubuchwepheshe be-pyrosequencing ukuthola emtholampilo ukuguqulwa kwe-KRAS kumdlavuza obala ngokombala, ngokunemba nokuthembeka okuhle. Le ndlela inokucaciswa okungcono nokuzwela okuphezulu. USundstrÖm et al. Uma kuqhathaniswa i-PCR eqondene ne-allelic kanye ne-pyrosequencing kuzicelo zomtholampilo futhi ithole ukuthi ezimweni ezingama-314 zokuguqulwa kwe-KRAS ezigulini ezinomdlavuza ezibalaseleyo, ukucaciswa kwe-pyrosequicing kwakuphakeme kunokwama-alleles. I-PCR, futhi inokuzwela okuhle kwizicubu ezinokuqukethwe kwamangqamuzana wesisu esincane. Nciphisa inani lamangqamuzana wesimila abe ngu-1.25% kuye ku-2.5%. I-Pyrosequigation isengathola amasiginali wokuguqula. Lapho okuqukethwe okuncane kwe-mutants alleles kusampula kudinga ukufinyelela kuma-20% ukuze kutholakale ngokulandelana kweSanger, kungatholwa ngendlela ye-HRM lapho ifinyelela ku-10%, futhi ekutholeni i-pyrosequicing kuphela ukuguqulwa okungatholakala nge-5%. Izindondo. Sisebenzise i-pyrosequicing ukuthola ukuguqulwa kwe-KRAS ezigulini ezingama-717 ezinomdlavuza obala futhi sathola ukuthi imvamisa yokuguqulwa kwe-KRAS yayingu-40.9%. Izinga lokuguqula i-codon 12 lalingu-30.1%, izinga lokuguqulwa kwekhodi engu-13 lalingu-9.8%, kanti izinga lokuguqulwa kwekhodi engu-61 lalingu-1.0%. Sicebise izicubu ngokuqukethwe okuphezulu kwesimila nge-manual microdissection ngaphambi kokuhlolwa, okwenza imiphumela ithembeke kakhulu. Le ndlela inokuzwelana okuhle nokucaciswa, futhi kulula ukuyithuthukisa ekwenzeni imitholampilo. Ukungahambi kahle kwe-pyrosequencing yizindleko eziphezulu zokutholwa, futhi inqubo yokulungisa i-DNA eyodwa-strand yokulandelana kwamasampuli inzima. Ngokuzayo, i-pyrosequencing inganikelwa ekuthuthukiseni ubuchwepheshe bokubona ngqo imikhiqizo ye-PCR esontwe kabili, okuzokwenza kube lula ukusebenza. Futhi wehlise ngempumelelo izindleko zokulandelana ukufeza ukukhuthazwa okuphelele kokuhlolwa kwemitholampilo.
Indlela ye-ARMS:
Lobu buchwepheshe busebenzisa ama-primers ukuhlukanisa phakathi kohlobo lwasendle nofuzo oluguqulwayo, wh
ich kuye kwabikwa ngawo-1980. Inzuzo enkulu yale ndlela ukuthi inokuzwela okufika ku-1.0% futhi ingathola izakhi zofuzo eziguqukayo kumasampuli aphansi njengo-1.0%. Ekuklanyeni, ubude bomkhiqizo oqondisiwe bungancishiswa kakhulu, futhi inkinga yokuthi imiphumela yokutholwa enembile ayikwazi ukutholwa ngoba iningi le-DNA ekhishwe kusifanekiso sesikhumba esifakwe kuphalafini ihlukene phakathi. Lobu buchwepheshe buhlanganisa ipulatifomu yesikhathi sangempela se-PCR ukufeza ukusebenza kwamashubhu avaliwe ngesikhathi sokukhulisa. Umsebenzi ulula futhi awudingi ukucubungulwa kwangemuva komkhiqizo, okungagwema ukungcoliswa komkhiqizo okhulisiwe ngezinga elikhulu kakhulu. Njengamanje, indlela ye-scorpion-ARMS ehlanganisa i-scorpion probe kanye ne-amplification block mutation system isetshenziswa kakhulu emhlabeni. Inhlanganisela yobuchwepheshe obubili ingakhulisa ukuzwela nokucaciswa kwezinhlangothi zombili. UGao Jie et al. Usebenzise le ndlela ukuthola isimo sokushintshwa kofuzo ku-KRAS ezigulini eziyi-167 ezinomdlavuza obala, okuphakamisa ukuthi le ndlela inokwethenjelwa futhi inembile. UWang Hui et al. Kusetshenziswe futhi i-ARMS ukuthola ukuguqulwa kwe-KRAS ezimweni eziyi-151 zezicubu ezi-formaldehyde-fixed kanye nophalafini. E-United States, ikhithi ye-COBAS (Roche) evunyelwe yi-FDA yokuhlolwa komtholampilo kwe-KRAS kanye neTherascreen RGQ kit (Qiagen) eqinisekiswe yi-European Union In Vitro Diagnostics (CE-IVD) bonke basebenzisa umgomo we-ARMS. Phakathi kwezindlela ezijwayelekile, indlela ye-ARMS iyabucayi kakhulu futhi izindleko zibiza kahle. Ngakho-ke, ingxenye enkulu yokutholwa kwemitholampilo yezakhi zofuzo ze-KRAS ekhaya nakwamanye amazwe isebenzisa indlela ye-ARMS, kepha ngenxa yokuthi le ndlela isuselwe kubuchwepheshe be-PCR, ukushiyeka kwayo ukuthi kungatholakala kuphela ukuguqulwa kwesayithi okwaziwayo.
4.Indlela ye-PCR yesilinganiso sangempela se-fluorescence:
It is a PCR-based detection method to determine the mutation by Ct value. It has the advantages of strong specificity, high sensitivity, accurate quantification, easy operation, and fully closed reaction. Many experimental groups have adopted this method for the detection of KRAS mutations in colorectal cancer. Compared with the direct sequencing method, quantitative PCR occupies a greater advantage in sensitivity. Most scholars comparing the two methods believe that quantitative PCR is more sensitive. Liu Wei et al. Used two methods to make a detailed analysis of the detection results of 280 cases of colorectal cancer KRAS gene mutations, 94 cases of KRAS gene sequencing mutations, the positive rate was 33.57% (94/280), of which, real-time fluorescence quantitative PCR was positive 91 cases had a sensitivity of 96.8% (91/94). Of the 186 gene sequencing wild-type cases, 184 were negative by real-time quantitative PCR, with a specificity of 98.9% (184/186). The coincidence rate between real-time fluorescence quantitative PCR method and direct gene sequencing method was 98.2%. In the two detection methods, the positive and negative coincidence rates of each mutation site were above 90%, and the coincidence rate of four sites reached 100%. The detection results of the two methods were highly consistent, indicating fluorescent quantitative PCR It is a more reliable method for mutation detection. However, PCR-based methods need to design primers and probes based on known mutation types, so all possible mutations cannot be detected, and only specific sites can be detected. If a certain site is not included in the detection range of the kit, even if there is actually a mutation, the kit result is still negative. In addition, although the sensitivity of quantitative PCR is high, whether there are false positives still needs to be verified by DNA sequencing technology, or retrospective and prospective clinical experiments with large sample sizes to confirm the correlation between KRAS mutation status and the efficacy of targeted drugs . Therefore, the high sensitivity of mutation detection should not be pursued blindly, while the specificity and accuracy of detection should be ignored. Under different laboratory conditions, the optimal method for mutation detection in specimens may also be different. For specimens with a higher proportion of mutations, Sanger sequencing method has a higher accuracy in detecting gene mutations, while for specimens with a lower proportion of mutations, Sanger sequencing method False negatives may occur, and the detection method using fluorescent PCR as the technical platform can be characterized by high sensitivity.
Indlela ye-HRM:
Ingenye yezindlela ezisetshenziswa kakhulu zokuthola izakhi zofuzo eminyakeni yamuva. Inezinzuzo zethubhu elula, esheshayo, ebucayi, neyodwa ukugwema ukungcoliswa. Ukuze kubhekwe ukuthi kungenzeka yini ukusetshenziswa kwayo ekuhlolweni kwemitholampilo, uLiu Liqin nabanye basebenzise indlela ye-HRM ukuthola ukuguquka kofuzo kwe-KRAS ezigulini ezingama-64 ezinomdlavuza obala, base besebenzisa ukulandelana okuqondile ukuqinisekisa imiphumela. Imiphumela ye-HRM nokulandelana okuqondile kutholakala kungaguquguquki. Uma kuqhathaniswa nokulandelana okuqondile, ukutholwa kwezinguquko zofuzo ze-KRAS yi-HRM kulula futhi kunembile, okuphakamisa ukuthi kuyindlela ethembekile efanelekile yokuhlolwa komtholampilo. UChen Zhihong et al. Kusetshenziswe indlela ye-HRM ukuhlola uchungechunge lwamasampuli axubekile aqukethe izilinganiso ezahlukahlukene zama-plasmids we-KRAS mutant ukuhlola ukuzwela kwawo. Kutholakale ukuthi inani lezinguquko ze-plasmid kumasampuli ahlanganisiwe lalingu-10%, futhi ukuzwela kwafinyelela ku-10%. Ngemuva kwalokho, le ndlela isetshenziselwe ukuthola ukuguqulwa kofuzo kuma-KRAS kumasampula wezicubu zomdlavuza ezingama-60. Uma kuqhathaniswa nendlela yokulandelana ngqo, ukuzwela kwendlela ye-HRM kwakungu-100%, futhi ukucaciswa kwakuyi-96% (43/45). Ububi bendlela ye-HRM ukuthi akunakwenzeka ukuhlinzeka ngokunembile uhlobo oluthile lokuguqula nokuthi iyiphi i-codon eshintshiwe. Uma kutholakala okungajwayelekile ejikeni lokuncibilika, indlela yokulandelana iyadingeka ukunquma uhlobo lokuguquka. Iqembu labacwaningi bakaHarlé lisebenzise amacala we-156 wezicubu zomdlavuza wokuqhathanisa imibala ukuqhathanisa i-PCR ye-fluorescent, izindlela ze-ARMS ne-HRM. Imiphumela ikhombisa ukuthi yize lezi zindlela ezintathu zikulungele ukuhlolwa kwemitholampilo, ukwethembeka kwe-HRM akukuhle njengezinye izindlela ezimbili.
Ezinye izindlela:
Ngaphezu kwezindlela ezibalulwe ngenhla, ezinye izindlela zokubona zinezinzuzo nezinkinga zazo ekusetshenzisweni, njenge-PCR-SSCP, ukusebenza okuphezulu kwe-chromatography, indlela ye-fluorescent PCR eyenziwe kahle ye-oligonucleotide probe, Indlela ye-PCR ehlanganisiwe kanye nenhlanganisela ye-ARMS, i-COLD-PCR indlela, njll. Ukusebenza okuphezulu kwe-chromatography ketshezi kunemininingwane eqinile, kepha isidingo samasampuli sikhulu; I-PCR-SSCP ibiza kancane futhi iyonga, kepha ukusebenza kuyinkimbinkimbi; ubuchwepheshe bokutholwa kokuguquka kwesisekelo bususelwa ku-PCR ye-fluorescent bunokucaciswa okuqinile, ukuzwela okuphezulu, nobuningi obunembile, Ukusebenza okulula, ukusabela okuvinjelwe ngokuphelele nezinye izinzuzo, kepha konke kudinga ukuklama izinto zokuqala neziphenyo ngokuya ngohlobo lokuguqula uhlobo olwaziwayo, ngakho-ke amasayithi athile kuphela kutholakele, futhi konke ukuguquka kwezinguquko okungenzeka kungatholakali.
I-3. Isifingqo
Ngokufingqa, ngoba amasayithi okuguqula izakhi zofuzo nezindlela zokubona kumalabhorethri ahlukene awafani, ubukhulu bezinhlobo zesimila ezihlaziyiwe kanye nekhwalithi yokukhishwa kwe-DNA nakho akulingani, okuholele ekutheni kube nemiphumela emikhulu noma emincane yokuhlola phakathi kokwehlukana kwamalabhorethri Ukutholwa kokuguqulwa kwezakhi zofuzo kwe-KRAS sekuyinkinga yokutholwa kwemitholampilo yokukhathazeka emazweni ahlukahlukene. Njengamanje, kunezindlela eziningi zokuthola ukuguqulwa kwezakhi zofuzo ku-KRAS. Ukuzwela kusuka phezulu kuye phansi yi-ARMS, i-pyrosequencing, i-HRM, i-PCR yokulinganisa yesikhathi sangempela, nokulandelana okuqondile. Kusuka eqinisweni lomtholampilo, ukuzwela okuphansi akuhambisani nokwelashwa kwemitholampilo, kepha izindlela ezibucayi kakhulu zingadala ukuthi ukutholwa kutholakale kwehle, futhi imiphumela emihle yamanga engadingekile ingenzeka futhi ithinte umuthi wezokwelapha olandelayo. Uma kubhekwa lezi zici ezingenhla, kuhlanganiswe nendlela evunyelwe yi-FDA, kunconywa indlela ye-ARMS. Vele, ngokubuka kwemakethe, ukuxilongwa kwamangqamuzana akufanele kungigcizelele
kodwa gxila emiphumeleni yokugcina efanele. Amalabhorethri ahlukene angasebenzisa izindlela zokuhlola ezifanele ngokuya ngesimo sangempela, kodwa kuphela uma eneziqu ezinhle zabasebenzisi kanye nezinhlelo zokulawula ikhwalithi zangaphakathi. Ngaphansi kwezimo zemvelo zamanje zaselabhorethri yasekhaya, kuyadingeka ukwenza ukuhlolwa elabhorethri ye-PCR esezingeni futhi ubambe iqhaza emisebenzini yokulawula ikhwalithi yasekhaya neyamazwe ngamazwe ukuze kuqinisekiswe ikhwalithi yokuhlola yaselabhorethri ethembekile. Ukuphatha okujwayelekile kuyisimo esidingekayo ukuze kuqinisekiswe imiphumela eqhubekayo. E-China, kunesidingo esiphuthumayo sokuhlanganisa kanye nokumisa ukuhlolwa komtholampilo kofuzo lwe-KRAS, kanye nokwenza uhlelo lokuhlola olusezingeni futhi olujwayelekile ngokwezidingo ezahlukene, futhi lolu hlelo lunganwetshwa ukuze kutholwe i-BRAF, PIK23450_3CA, EGFR kanye ezinye izakhi zofuzo ukuthuthukisa ukuhlolwa kwe-pathology yamangqamuzana emtholampilo.
