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. Iyo yakanaka mwero weKRAS gene mutation mune colorectal cancer
Mune kenza yakajeka, mwero wekuchinja weiyo KRAS geni yakakwira kunge 35% kusvika 45%, uye nzvimbo ine njodzi huru yekuchinja nzvimbo macodon 12 ne13 pane exon 2, uye kuchine zvisingawanzo zvakaita se61 ne146. saiti. Kune nzira dzakawanda dzekucherechedza kweKRAS gene shanduko, kusanganisira kuenzanirana, kukwirisa kwakanyanya kunyungudika curve kuongorora (HRM), pyrosequencing, huwandu hwePCR, mutation amplification block system (amplinc atio) nrefractorymutation system (ARMS), yekudzivirira chidimbu kureba kwepolymorphism (RFLP), polymerase cheni reaction-single-strand conformation polymorphism ongororo (PCR-singlestrand confomation polymorphism (PCR-SSCP), co-amplification pazasi denaturation temperatur PCR (COLD-PCR) uye yakakwira-kuita kwemvura chromatography ongororo, nezvimwe.
2. Kuongororwa kweKRAS nzira dzekushandurwa kwekuchinja
1. Yakananga nzira yekuteedzera: Ndiyo nzira yepamusorosoro yekuona KRAS geni shanduko, uye zvakare iri yegoridhe chiyero chekuona kushanduka kwemajini. Iyo yakanangana nzira yekuteedzera inoenderana nesisitimu yeiyo dhiyoxy kuteedzana inogona kuratidza intuitively kuratidza shanduko yekuteedzana kwemajini muchimiro chepasi peepu. Rudzi rwekutsvaga rwakanyanya kuwedzeresa, uye zvakare ndiyo yekutanga nzira inoshandiswa yekuchinja maitiro. Kunyangwe paine kubuda kwechizvarwa chitsva chekuteedzera mapuratifomu, vadzidzi vekumba nekune dzimwe nyika vachiri kushandisa mhedzisiro yekuteedzera kwakaringana seyero yekuyera uye kuona kuvimbika kwenzira nyowani. Gao Jing et al. Inoshandiswa yakateedzana yakanangana nekuonekwa kweKRAS uye BRAF geni shanduko mune varwere 966 vane cancer yakajeka. Uku ndiko zvakare kuongorora kweiyo KRAS gene mutation pamwe neyakakura sampuro yemumba yakataurwa mumabhuku. Ling Yun nevamwe vanotenda kuti nzira yakanangana nekuteedzera ndiyo yakanangana uye inoshanda nzira yekutsvaga yekunzwisisa mamiriro ekuchinja kwejena rega, iro rinogona kujekesa mhando yeshanduko, kunyanya pakuonekwa kwekuchinja kusingazivikanwe. Kunyangwe kunzwisisika kwenzira iyi kwakadzikira, kunogona kuvandudzwa nenzira senge microdissection yekuvandudza mamota maseru. Iyo yakanangana nzira yekuteedzera yakashandiswawo kune iyo KRAS kuona kweakakura sampuro saizi mune mamwe mapoka epamba ekutsvagisa. Nekudaro, kuderera kwekunzwa ndiko kusakosha kukuru kwekuteedzera kwakanangana. Tichitarisa kubva kumhedzisiro yakataurwa kuChina, iyo mwero yekucherechedzwa kwehuwandu nekuteedzera kwakanangana haisi yakaderera. Liu Xiaojing et al. Inofananidzwa yakateedzana yakateerana uye peptide nucleic acid simbi PCR (PNA-PCR) uye ndokuona kuti makumi mana nematatu emakesi eKRAS gene magene akaonekwa nekuteedzera kwakanangana. Pamusoro pekuchinja uku, PNA-PCR yakaonekwawo nekuteedzera kwakanangana. Shanduko gumi dzakawanikwa mumhando yemusango, uye mazano akaitwa ekuti uone mhando yemusango varwere nePCR uye yakanangana nzira yekuteedzera kuti vaone varwere vanochinja. Qiu Tian et al. Yakaonekwa 131 colorectal cancer specimens ne fluorescent PCR-yakagadziriswa oligonucleotide probe nzira uye yakanangana nzira yekuteedzera, uye iwo mazinga akanaka eKRAS gene magene aive 41.2% (54/131) uye 40.5% (53/131)). Bai Dongyu akakurukurawo nezvekuonekwa kwekunzwa kwenzira dzakasiyana. Pakati pevarwere vegomarara vane mazana maviri nemazana maviri, makumi matanhatu nemapfumbamwe vakaonekwa neRT-qPCR shanduko, uye mwero wekuonekwa kwekuchinja waiva 200%; 63 sampuli dzakateedzana zvinobudirira nekuteedzera kwakanangana makumi mashanu emakesi ekuchinja, shanduko yekuona mwero 31.5%. Kunyangwe iyo nzira yekuteedzera yakanangana inogona kunyatso, yakajeka uye yakanangana kuona iyo KRAS gene mutation chinzvimbo, zvikanganiso zvayo senge zvakakwirira zvehunyanzvi zvinodiwa, yakaoma mashandiro maitiro, zviri nyore kukonzera kusvibiswa kwepamberi, uye inopedza nguva uye kududzira kunonetsa kwemhedzisiro zvakare pachena. Kazhinji hapana michina yekuteedzana, uye iyo specimen inoda kutumirwa kune inoenderana nekambani kunoedzwa, iyo inotora nguva yakareba uye ine mutengo wakakwira, saka ine zviyero zvakakura.
Pyrosequencing nzira:
Pyrosequicing nzira zvakare iri nyore nzira yeKRAS yekuchinja kwemajini kuona maererano nekuteedzana kwekunzwa, kuona mutengo uye nguva yekumhan'ara. Kudzokorora kweiyi nzira kuri nani. Zvinoenderana neyakawanikwa yepamusoro mepu Kuverengera kudzidza kwehuwandu hwekuchinja kweimwe saiti uye kuenzanisa pakati penzvimbo dzekuchinja kwenzvimbo dzakasiyana kwakajeka pakatarisa. Mumakore apfuura, Ogino et al., Hutchins et al. Washandisa pyrosequencing tekinoroji kuyedza shanduko dzeKRAS mune varwere vane yakakura masampula ekenza yakajeka. Mhedzisiro yacho inoratidza kuti pyrosequencing tekinoroji chishandiso chine simba chekutarisa varwere kurapa kwakanangwa. Tumor mamorekuru kuongororwa ane yakafara application tarisiro. Nyanzvi dzemudzimba dzakashandisawo pyrosequencing tekinoroji kukiriniki kuona shanduko yeKRAS mukenza yakajeka, nekururama kwakanaka uye kuvimbika. Iyi nzira ine zviri nani kujekesa uye kukwirira kwekunzwa. SundstrÖm et al. Inofananidzwa ne-allelic-yakatarwa PCR uye pyrosequencing mumakiriniki ekushandisa uye akawana kuti muzviitiko mazana matatu nemakumi mana nezvina zvekuchinja kweKRAS muvarwere vegomarara, iko kusarudzika kwepirose kwaive kwakakwirira kupfuura kwea alleles. PCR, uye ine kugona kwakanaka kumatukisi ane yakaderera bundu maseru zvemukati. Deredza chiyero chemarara maseru kusvika 314% kusvika 1.25%. Pyrosequencing inogona kuona masayendisiti ekuchinja. Kana zvirinyore zvemutant alleles mumuenzaniso zvinoda kusvika makumi maviri kubva muzana kuti zvionekwe neSanger kuteedzana, zvinogona kuonwa nenzira yeHRM kana yasvika gumi muzana, uye pyrosequicing chete shanduko dzinogona kuwanikwa ne2.5%. Alleles. Isu takashandisa pyrosequicing kuona KRAS mutations mune 20 varwere vane colorectal cancer uye takaona kuti kuwanda kweKRAS shanduko yaive 10%. Iyo mwero wekuchinja wecodon 5 yaive 717%, iyo shanduko mwero wekodhi 40.9 yaive 12%, uye mwero wekuchinja wecodon 30.1 yaive 13%. Isu takafumisa iwo matukisi neakakwira bundu zvemukati neicho bhuku microdissection usati waedzwa, tichiita kuti mhedzisiro iwedzere kuvimbika. Iyo nzira ine kugona kwakanaka uye kusarongeka, uye zviri nyore kukudziridza mune yekiriniki tsika. Iko kusagadzikana kweiyo pyrosequencing ndiyo yakakwira mutengo wekutsvaga, uye maitiro ekugadzirira imwechete-yakashama DNA yekuteedzera masampula inonetsa. Mune ramangwana, pyrosequencing inogona kuiswa mukugadzirwa kwetekinoroji yekutarisa yakananga yeviri-yakashomeka PCR zvigadzirwa, izvo zvicharerutsa mashandiro acho. Uye zvinobudirira kudzikisa mutengo wekuteedzera kuti uwane zvakakwana kusimudzira kwekuongororwa kwekiriniki.
3. MaARMS nzira:
Iyi tekinoroji inoshandisa primers kusiyanisa pakati pesango-mhando uye mutant majini, wh
ich yakataurwa kare kuma1980. Kubatsira kukuru kweiyi nzira ndeyekuti ine kunzwisisika inosvika ku1.0% uye inogona kuona majini anoshanduka mumasampuli akadzika se1.0%. Mukugadzirwa, kureba kwechigadzirwa chakanangwa kunogona kupfupiswa kusvika padanho rakakura, uye dambudziko rekuti mhedzisiro yekutsvaga chaiyo haigone kuwanikwa nekuti yakawanda yeDNA yakabviswa muparafini-yakadzamirwa yemucherechedzo wetissue yakatsemurwa. Iyi tekinoroji inosanganisa iyo chaiyo-nguva PCR chikuva kuti iite yakavharwa-chubhu mashandiro panguva yekuwedzeredza. Iko kuvhiya kuri nyore uye hakudi kuti post-kugadzirisa kwechigadzirwa, icho chinogona kudzivirira kusvibiswa kwechigadzirwa chakawedzerwa kusvika pamwero mukuru. Parizvino, iyo scorpion-ARMS nzira inosanganisa scorpion probe uye amplification block mutation system inowanzo shandiswa pasi. Iko kusanganiswa kwetekinoroji mbiri kunogona kuwedzera kusanzwisisa uye kusarudzika kwemativi ese. Gao Jie et al. Vakashandisa nzira iyi kuona iyo KRAS geni mutation chinzvimbo mune 167 varwere vane colorectal cancer, zvichiratidza kuti nzira iyi yakavimbika uye yakarurama. Wang Hui et al. Zvakare yakashandiswa maARMS kuona KRAS shanduko mune 151 kesi dze formaldehyde-yakagadziriswa uye parafini-yakadzikwa matishu. MuUnited States, iyo COBAS kit (Roche) yakabvumidzwa neDFA kuongororwa kwekiriniki kweKRAS uye neTrascreen RGQ kit (Qiagen) yakasimbiswa neEuropean Union In Vitro Diagnostics (CE-IVD) vese vanoshandisa mutemo weARMS. Pakati penzira dzinojairika, nzira yeARMS ndiyo yakanyanya kuomarara uye mutengo wacho unonzwisisika. Naizvozvo, chikamu chikuru chekiriniki yekuonekwa kwemajini eKRAS kumba nekune dzimwe nyika vari kushandisa nzira yeARMS, asi nekuti nzira yacho yakavakirwa paPCR tekinoroji, kukanganisa kwayo ndeyekuti inogona kungoonekwa Inozivikanwa saiti shanduko.
4. Real-time fluorescence yakawandisa PCR nzira:
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.
5. Nzira yeHRM:
Iyo ndeimwe yedzinowanzo shandiswa nzira dzekutsvaga kwemajini mumakore apfuura. Iine zvakanakira yakapusa, inokurumidza, inonetsekana, uye imwechete chubhu kudzivirira kusvibiswa. Kuti uongorore kugona kwekushandisa kwayo mukuyedzwa kwekiriniki, Liu Liqin nevamwe vakashandisa nzira yeHRM kuona KRAS gene magene muvarwere makumi matanhatu nevatanhatu vane gomarara rakakora, ndokuzoshandisa kuteedzana kwakanangana kuti vaone zvabuda. Mhedzisiro yeHRM uye yakanangana nekuteedzana inoonekwa seyakaenzana. Zvichienzaniswa nekuteedzera kwakanangana, kuonekwa kweKRAS gene maNHR kuri nyore uye kwakaringana, zvichiratidza kuti inzira yakatendeka inokodzera kuyedzwa kwekiriniki. Chen Zhihong et al. Akashandisa nzira yeHRM kuyedza akateedzana masampuli akasanganiswa ane huwandu hwakasiyana hweKRAS mutant plasmids kuongorora kwavo kunzwisisika. Izvo zvakawanikwa kuti chikamu cheplasmid shanduko mune yakasanganiswa sampuli yaive 64%, uye kunzwisisika kwakasvika 10%. Shure kwaizvozvo, nzira yacho yaishandiswa kuona KRAS gene magene mu10 colorectal cancer cancer sampuli. Zvichienzaniswa neyakanangana nenzira yekuteedzana, kunzwisisika kwenzira yeHRM yaive 60%, uye iyo yakatarwa yaive 100% (96/43). Iko kusakosha kwenzira yeHRM ndeyekuti hazvigoneke kunyatso kupa iyo chaiyo mhando yekuchinja uye iyo codon inoshandurwa. Kana kusanzwisisika kukawanika pane iyo yakanyungudika curve, nzira yekuteedzana inodiwa kuti uone iyo mutation mhando. Boka rekutsvagisa reHarlé rakashandisa 45 kesi dzecolorectal cancer turu kuenzanisa fluorescent PCR, ARMS uye HRM nzira. Mhedzisiro yacho inoratidza kuti kunyangwe nzira nhatu dzakakodzera kuyedzwa kwekiriniki, kuvimbika kweHRM hakuna kunaka sedzimwe nzira mbiri.
6. Dzimwe nzira:
Pamusoro penzira dzataurwa pamusoro apa, dzimwe nzira dzekutsvaga dzine zvadzinobatsira pamwe nezvazviri mukushandisa, senge PCR-SSCP, yakanyanya kuita chromatography yemvura, fluorescent PCR-yakagadziriswa oligonucleotide probe nzira, Maitiro edendere PCR neARMS mubatanidzwa, COLD-PCR nzira, nezvimwewo. High performance liquid chromatography ine kusimba kwakasimba, asi kudiwa kwemasampuli kwakakura; PCR-SSCP yakaderera mumutengo uye ine mari, asi mashandiro acho akaomarara; iyo mutation yekuona tekinoroji yakavakirwa pane fluorescent PCR ine kusimba kwakasimba, kukwirira kwakanyanya, uye hwakaringana huwandu, Easy oparesheni, yakavharwa zvakakwana kuita uye zvimwe zvakanakira, asi zvese zvinofanirwa kugadzira primers uye probes zvinoenderana nerinozivikanwa shanduko mhando, saka chete chaiwo masosi anogona kuve yakaonekwa, uye zvese zvingangoitika shanduko hazvigone kuwanikwa.
3. pfupiso
Mukupfupisa, nekuti nzvimbo dzekuchinja uye nzira dzekutsvaga mumarabhoritari akasiyana hadzisi yunifomu, saizi yemamota akaongororwa uye hunhu hwekutorwa kweDNA hahuna kuenzana, zvichikonzera kuvepo kwemhedzisiro yakakura kana diki yekuyedza pakati pemarabhoritari Misiyano. ye KRAS gene mutation detection yave kuita kiriniki yekutsvaga nyaya yekunetseka munyika dzakasiyana siyana. Parizvino, kune nzira dzakawanda dzekuona shanduko mujeni reKRAS. Iko kunzwisisika kubva kumusoro kusvika kuzasi maARMS, pyrosequencing, HRM, chaiyo-nguva yakawandisa PCR, uye yakanangana nekuteedzana. Kubva kuicho chaihwo chemakiriniki, kushomeka kushoma hakukodzere kurapwa, asi nzira dzakaomarara dzinokonzeresa kuti huzivi huderere, uye mhedzisiro isingadiwe yenhema inogona kuitika ichikanganisa maringerenzi emurwere anotevera. Tichifunga nezve zviri pamusoro zvinhu, zvakasanganiswa nenzira yakatenderwa neDFA, nzira yeARMS inokurudzirwa. Ehezve, kubva pamusika maonero, mamorekuru kuongororwa haafanire kundisimbisa
mitsetse, asi tarisa pane yekupedzisira chaiyo mhinduro. Marabhoritari akasiyana anogona kutora nzira dzekuyedza dzakakodzera zvinoenderana nemamiriro chaiwo, asi chete kana aine dhizaini dhizaini uye yemukati yemhando yekudzora masisitimu. Pasi pemamiriro azvino emurabhoritari yemamiriro ekunze, zvinodikanwa kuita bvunzo mune yakajairwa PCR murabhoritari uye kutora chikamu mune zvemukati uye zvepasi rose zvemukati-makamuri ekudzora mhando zviitiko kuti ive nechokwadi chekuvimbika murabhoritari yekuyedza mhando. Standardized manejimendi chinhu chinodiwa kuti uve nechokwadi chenguva dzose mhedzisiro. MuChina, pane kudiwa kwekukurumidzira kubatanidza uye kuenzanisa kuongororwa kwekiriniki yeKRAS gene, uye kugadzira chirongwa chakamisikidzwa uye chakamisikidzwa chekuyedza zvinoenderana nezvinodiwa zvakasiyana, uye chirongwa ichi chinogona kuwedzerwa kusvika pakuonekwa kweBRAF, PIK23450_3CA, EGFR uye mamwe majini ekusimudzira kiriniki yemamolecular pathology yekuongorora.
