An yi amfani da magungunan da aka yi niyya kamar su cetuximab da panituumab a cikin asibiti a matsayin ingantattun magungunan warkewa don ciwon daji na colorectal. Bayanai na asibiti sun nuna cewa marasa lafiya tare da maye gurbin KRAS ba su da wani tasiri mai mahimmanci akan wannan maganin rigakafin ƙwayar cuta na monoclonal, kuma marasa lafiya na daji ne kawai za su iya amfana da shi. Sabili da haka, ana ɗaukar matsayin maye gurbin kwayoyin halitta na KRAS a asibiti a matsayin muhimmiyar alamar warkewa, wanda ke da alaƙa mai ƙarfi tare da tsinkaya da tasirin maganin ciwon daji. Jagororin Ayyukan Clinical Clinical Cancer na 2009 na ƙasa (NCCN) sun nuna cewa duk majinyata da ke da ciwon daji na metastatic dole ne su gano yanayin maye gurbin halittar KRAS, kuma nau'in daji na KRAS kawai ana ba da shawarar karɓar maganin EGFR da aka yi niyya. A wannan shekarar, al'ummar likitocin asibiti na Amurka (ASCO) ta bayar da shawarwarin magani iri daya a matsayin alamar kwayoyin halitta don taki da yawa, wanda ke nuna mahimmancin mahimmancin mahimmanci. A halin yanzu, an gudanar da gwajin kwayoyin halitta na KRAS a asibiti. Mu galibi muna kimanta hanyoyin gano maye gurbi na KRAS na cikin gida don tunani cikin zaɓin asibiti.
1. Matsayi mai kyau na maye gurbin kwayar cutar KRAS a cikin cutar sankarau
A cikin cutar sankarau, yanayin maye gurbi na kwayar halittar KRAS ya kai 35% zuwa 45%, kuma rukunin maye gurbi mai haɗari shine codons 12 da 13 akan exon 2, kuma har yanzu akwai waɗanda ba kasafai ake samun irin su 61 da 146 ba shafin. Akwai hanyoyin ganowa da yawa don maye gurbi na KRAS, gami da jerawa kai tsaye, babban bincike mai narkewa (HRM), kayyadadden sakamako, PCR mai yawa, tsarin toshewa na maye gurbi (amplinc atio) nrefractorymutation system (ARMS), ƙayyadadden guntun yanki polymorphism (RFLP), polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-singlestrand confomation polymorphism (PCR-SSCP), haɓakawa tare a ƙananan denaturation temperatur PCR (COLD-PCR) da haɓakar aikin chromatography mai ƙarfi, da dai sauransu.
2. Kimar hanyoyin gano maye gurbi na KRAS
1. Hanyar jeran hanya kai tsaye: Hanya ce mafi dacewa don gano maye gurbi na KRAS, kuma shima mizanin zinare ne don gano maye gurbi. Hanyar jeran kai tsaye bisa dogaro da tsarin dideoxy jerawa zai iya nuna canjin hankali sau da ƙira cikin sifar taswirar tushe. Na'urar ganowa ta fi cikakke, kuma ita ce hanyar gano maye gurbi da farko. Duk da bullar sabbin dandamali na tsara tsara, amma har yanzu masana a cikin gida da kasashen waje suna amfani da sakamakon tsarin kai tsaye a matsayin sikeli don auna da kuma tabbatar da amincin sabuwar hanyar. Gao Jing et al. Anyi amfani da jerin tsararru kai tsaye don gano maye gurbi na KRAS da BRAF a cikin marasa lafiya 966 masu cutar kansa. Wannan kuma bincike ne na maye gurbi na KRAS tare da mafi girman samfurin gida da aka ruwaito a cikin adabi. Ling Yun da wasu sun yi imani da cewa hanyar jerawa kai tsaye ita ce hanya mafi tsada kuma mafi inganci don fahimtar yanayin rikidar kowane jinsi, wanda zai iya fayyace nau'in maye gurbi, musamman don gano maye gurbi da ba a sani ba. Kodayake ƙwarewar wannan hanyar ba ta da sauƙi, ana iya inganta ta hanyoyi kamar microdissection don wadatar da ƙwayoyin tumo. Hakanan an yi amfani da hanyar jerin tsararru kai tsaye zuwa ga binciken KRAS na manyan samfuran samfuran a cikin wasu ƙungiyoyin bincike na cikin gida. Koyaya, ƙananan ƙwarewa shine babbar rashin dacewar jerantawa kai tsaye. Idan aka yi la'akari da sakamakon da aka ruwaito a cikin Sin, ƙimar gano maye gurbi ta hanyar jerawa kai tsaye ba ƙasa bane. Liu Xiaojing et al. Idan aka kwatanta daidaito kai tsaye da peptide nucleic acid clamp PCR (PNA-PCR) kuma sun gano cewa an gano al'amuran 43 na maye gurbi na KRAS ta hanyar jerin kai tsaye. Baya ga waɗannan maye gurbi, an gano PNA-PCR ta hanyar jera kai tsaye. An samo maye gurbi goma a cikin nau'in daji, kuma an ba da shawarwari don ƙayyade marasa lafiyar nau'in daji ta hanyar PCR da kuma hanyar kai tsaye kai tsaye don ƙayyade marasa lafiyar. Qiu Tian et al. An gano nau'ikan 131 na cutar kansa ta hanyar mai binciken PC o-ingantaccen oligonucleotide da tsarin bin tsarin kai tsaye, kuma ingantattun matakan maye gurbi na KRAS sune 41.2% (54/131) da 40.5% (53/131)). Bai Dongyu ya kuma tattauna ƙwarewar gano hanyoyi daban-daban. Daga cikin 200 marasa lafiya masu cutar kansa, 63 an gano ta ta hanyar maye gurbin RT-qPCR, kuma yawan gano maye gurbin ya kasance 31.5%; Samfurai 169 anyi nasarar tsara su ta hanyar tsara kai tsaye sau 50 na maye gurbi, yanayin gano maye gurbi 29.6%. Kodayake hanyar jerin tsararru na iya daidai, da hankali kuma musamman gano yanayin maye gurbi na KRAS, gazawarsa kamar manyan buƙatun fasaha, hanyoyin aiki masu rikitarwa, sauƙin haifar da gurɓataccen gurbi, da cinye lokaci da fassarar sakamako mai ƙima. bayyananne. Sau da yawa babu kayan aikin jerawa, kuma samfurin yana buƙatar aikawa zuwa kamfanin da ya dace don gwaji, wanda ke ɗaukar lokaci mai tsawo kuma yana da tsada mai yawa, saboda haka yana da iyakoki masu yawa.
Hanyar Pyrosequencing:
Hanyar Pyrosequencing ita ma hanya ce mafi dacewa don gano maye gurbi na KRAS dangane da ƙididdigar ƙararrawa, tsadar ganowa da lokacin yin rahoto. Maimaitawar wannan hanyar ita ce mafi kyau. Dangane da tsararren taswirar da aka samo Nazarin adadi na yawan maye gurbi na wani shafin da kwatankwacin abubuwan canzawa na shafukan yanar gizo a bayyane yake. A cikin 'yan shekarun nan, Ogino et al., Hutchins et al. Yi amfani da fasaha na zamani don gwada maye gurbin KRAS a cikin marasa lafiya tare da manyan samfuran cutar kansa. Sakamakon ya nuna cewa fasaha na zamani shine kayan aiki mai ƙarfi don bincika marasa lafiya don maganin fargaba. Ciwon ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta tana da cikakkun damar aikace-aikace. Masana cikin gida sun kuma yi amfani da fasahar zamani don gano maye gurbi na KRAS a cikin cutar kansa, tare da kyakkyawan daidaito da aminci. Wannan hanyar tana da ƙayyadaddun bayanai da ƙwarewa mafi girma. SundstrÖm et al. Idan aka kwatanta da takamaiman PCR da takaddama a aikace-aikacen asibiti kuma sun gano cewa a cikin sharuɗɗan 314 na maye gurbi na KRAS a cikin masu fama da cutar sankarar hanji, ƙayyadadden abin da ya faru ya fi na alleles. PCR, kuma yana da ƙwarewa ga kyallen takarda tare da ƙarancin ƙwayar ƙwayar ƙwayar cuta. Tsarma rabo daga kwayoyin tumo zuwa 1.25% zuwa 2.5%. Pyrosequencing har yanzu yana iya gano siginar maye gurbi. Lokacin da mafi ƙarancin abun da ke tattare da mutant alleles a cikin samfurin yana buƙatar isa 20% don ganowa ta hanyar Sanger, ana iya gano shi ta hanyar HRM lokacin da ya kai 10%, kuma don ƙaddamarwa kawai maye gurbi zai iya ganowa da 5%. Allah. Munyi amfani da sakamako don gano maye gurbi na KRAS a cikin marasa lafiya 717 tare da ciwon daji na launi kuma mun gano cewa yawan maye gurbi na KRAS shine 40.9%. Tsarin maye gurbi na codon 12 ya kasance 30.1%, adadin maye gurbi na codon 13 ya kasance 9.8%, kuma adadin maye gurbin lambar codon 61 ya zama 1.0%. Mun wadatar da kyallen takarda tare da abun ciki na ƙari mafi girma ta hanyar microdissection na hannu kafin gwaji, muna mai da sakamakon ya zama abin dogaro. Hanyar tana da kyakkyawar fahimta da takamaiman abu, kuma yana da sauƙin ci gaba a aikin asibiti. Rashin dacewar lalacewar sakamako shine tsadar ganowa, kuma tsarin shirya DNA mai madaidaici don samin samfura yana da wahala. A nan gaba, za a iya sadaukar da kayyadadden sakamako ga ci gaban fasaha don gano kayayyakin PCR masu ruwa biyu, kai tsaye wanda zai saukaka aikin. Kuma yadda yakamata a rage kuɗaɗen jadawalin don samun cikakken ci gaba na gwajin asibiti.
3. Hanyar ARMS:
Wannan fasaha tana amfani da abubuwan share fage don rarrabewa tsakanin nau'in-daji da rikirkita kwayoyin halittu, wh
An bayar da rahoton ich tun farkon 1980s. Babban fa'idar wannan hanyar ita ce cewa yana da ƙwarewa har zuwa 1.0% kuma yana iya gano ƙwayoyin halittar mutant a cikin samfuran ƙasa da 1.0%. A cikin ƙira, za a iya taƙaita tsawon samfurin abin da aka sa gaba har zuwa mafi girma, kuma matsalar da ba za a iya samun cikakken sakamakon ganowa ba saboda yawancin DNA da aka ciro daga samfurin jikin paraffin ɗin ya kasu kashi biyu. Wannan fasaha ta haɗu da ainihin lokacin dandamalin PCR don cimma aikin ƙulli-ƙulli yayin haɓakawa. Aikin yana da sauki kuma baya buƙatar aikin bayan samfurin, wanda zai iya kauce wa gurɓatarwar samfurin da aka haɓaka zuwa mafi girman harka. A halin yanzu, hanyar kunama-ARMS da ke haɗuwa da binciken kunama da tsarin maye gurɓin haɓɓakawa ana amfani da ita a duniya. Haɗuwa da fasahohin biyu na iya haɓaka ƙwarewa da takamaiman ɓangarorin biyu. Gao Jie et al. An yi amfani da wannan hanyar don gano yanayin maye gurbi na KRAS a cikin marasa lafiya 167 da ke fama da cutar kansa, yana nuna cewa wannan hanyar abin dogaro ce. Wang Hui et al. Har ila yau, an yi amfani da ARMS don gano maye gurbi na KRAS a cikin sharuɗɗan 151 na formaldehyde-tsayayyen da kayan haɗin paraffin. A Amurka, kayan COBAS (Roche) waɗanda FDA ta amince da su don gwajin asibiti na KRAS da Therascreen RGQ kit (Qiagen) waɗanda Unionungiyar Tarayyar Turai In Vitro Diagnostics (CE-IVD) ta amince da su duka suna amfani da ƙa'idar ta ARMS. Daga cikin hanyoyin gama gari, hanyar ARMS ita ce mafi mahimmancin hankali kuma farashin yana da ƙima mai sauƙi. Sabili da haka, babban ɓangaren binciken asibiti na kwayoyin KRAS a cikin gida da ƙasashen waje suna amfani da hanyar ARMS, amma saboda hanyar ta dogara ne akan fasahar PCR, gazawarta shine cewa za'a iya gano shi kawai Sanadaran rukunin yanar gizo.
4. Hanyar PCR ta ainihin lokaci mai haske:
Hanya ce ta tushen PCR don tantance maye gurbi ta ƙimar Ct. Yana da fa'idodi na ƙayyadaddun ƙaƙƙarfan ƙaƙƙarfan ƙaƙƙarfan ƙayyadaddun hankali, babban azanci, ƙididdige ƙididdigewa, aiki mai sauƙi, da cikakkiyar amsawa. Ƙungiyoyin gwaji da yawa sun ɗauki wannan hanyar don gano maye gurbin KRAS a cikin ciwon daji na launin fata. Idan aka kwatanta da hanyar jeri kai tsaye, PCR mai ƙididdigewa yana da fa'ida mafi girma a cikin hankali. Yawancin malaman da ke kwatanta hanyoyin biyu sun yi imanin cewa PCR mai yawa ya fi kulawa. Liu Wei et al. An yi amfani da hanyoyi guda biyu don yin cikakken bincike game da sakamakon gano 280 na ciwon daji na launi na KRAS gene maye gurbi, lokuta 94 na maye gurbin kwayoyin halittar KRAS, ƙimar da ta dace ya kasance 33.57% (94/280), wanda, ainihin-lokacin da ake kira fluorescence quantitative. PCR yana da inganci 91 lokuta suna da hankali na 96.8% (91/94). Daga cikin nau'ikan nau'ikan nau'in daji na 186, 184 ba su da kyau ta hanyar PCR na ainihin lokaci, tare da ƙayyadaddun 98.9% (184/186). Matsakaicin daidaituwa tsakanin hanyar PCR mai saurin haske na ainihin-lokaci da hanyar jeri-biyu kai tsaye shine 98.2%. A cikin hanyoyin gano guda biyu, ƙimar daidaituwa mai kyau da mara kyau na kowane rukunin maye gurbi sun haura kashi 90%, kuma adadin rukunoni guda huɗu ya kai 100%. Sakamakon gano hanyoyin biyu sun yi daidai sosai, suna nuni da PCR mai kyalli Yana da ingantacciyar hanya don gano maye gurbi. Koyaya, hanyoyin tushen PCR suna buƙatar ƙirƙira ƙirar ƙira da bincike bisa sanannun nau'ikan maye gurbi, don haka ba za a iya gano duk maye gurbi ba, kuma takamaiman rukunin yanar gizo kawai za'a iya ganowa. Idan ba a haɗa wani rukunin yanar gizon a cikin kewayon gano kayan ba, ko da a zahiri akwai maye gurbi, sakamakon kit ɗin har yanzu mara kyau. Bugu da ƙari, ko da yake ƙwarewar PCR mai ƙididdigewa yana da girma, ko akwai ƙididdiga na ƙarya har yanzu yana buƙatar tabbatarwa ta hanyar fasahar jerin DNA, ko gwaje-gwajen gwaje-gwaje na asibiti na baya-bayan nan tare da manyan nau'in samfurin don tabbatar da daidaituwa tsakanin matsayi na maye gurbin KRAS da ingancin abin da aka yi niyya. kwayoyi . Don haka, bai kamata a bi diddigin yawan gano maye gurbi a makance ba, yayin da ya kamata a yi watsi da ƙayyadaddun ganowa da daidaito. A ƙarƙashin yanayin dakin gwaje-gwaje daban-daban, mafi kyawun hanyar gano maye gurbi a cikin samfura na iya bambanta. Don samfuran da ke da mafi girman adadin maye gurbi, hanyar bin diddigin Sanger yana da daidaito mafi girma wajen gano maye gurbi, yayin da samfuran da ke da ƙananan sauye-sauyen maye gurbi, hanyar bin diddigin Sanger Ƙarya na iya faruwa, da kuma hanyar ganowa ta amfani da PCR mai kyalli a matsayin dandalin fasaha. ana iya siffanta shi da babban hankali.
5. HRM hanya:
Yana daya daga cikin hanyoyin gano kwayoyin halitta da akafi amfani dasu a cikin recentan shekarun nan. Yana da fa'idodi na sauƙi, mai sauri, mai mahimmanci, da bututu ɗaya don kauce wa gurɓataccen abu. Don bincika yuwuwar amfani da shi a gwajin asibiti, Liu Liqin da sauransu sun yi amfani da hanyar HRM don gano maye gurbi na KRAS a cikin marasa lafiya 64 da ke fama da cutar sankarau, sannan suka yi amfani da tsararraki kai tsaye don tabbatar da sakamakon. Sakamakon HRM da jeranta kai tsaye an same su daidai. Idan aka kwatanta da jerawa kai tsaye, gano maye gurbi na KRAS ta HRM abu ne mai sauƙi kuma daidai, yana nuna cewa hanya ce abin dogaro wacce ta dace da gwajin asibiti. Chen Zhihong et al. Anyi amfani da hanyar HRM don gwada jerin nau'ikan samfuran gauraye waɗanda ke ƙunshe da nau'ikan daban-daban na KRAS mutant plasmids don kimanta ƙwarewar su. An gano cewa adadin maye gurbi na plasmid a cikin hadaddun samfuran 10% ne, kuma ƙwarewar ta kai 10%. Bayan haka, an yi amfani da hanyar don gano maye gurbi na KRAS a cikin 60 samfurin nama na kansar kai tsaye. Idan aka kwatanta da tsarin jeranta kai tsaye, ƙwarewar hanyar HRM ta kasance 100%, kuma takamaiman shine 96% (43/45). Rashin dacewar hanyar HRM shine cewa ba shi yiwuwa a samar da takamaiman nau'in maye gurbi kuma wane codon ne aka canza. Idan aka sami mummunan abu akan ƙwanƙwasa narkewa, ana buƙatar hanyar jerawa don ƙayyade nau'in maye gurbi. Researchungiyar bincike ta Harlé ta yi amfani da shari'o'in 156 na ƙwayar cutar kansa ta kwalliya don kwatanta hanyoyin PCR mai haske, ARMS da HRM. Sakamakon ya nuna cewa kodayake hanyoyin guda uku sun dace da gwajin asibiti, amincin HRM ba shi da kyau kamar sauran hanyoyin biyu.
6. Sauran hanyoyin:
Baya ga hanyoyin da muka ambata a sama, sauran hanyoyin ganowa suna da nasu fa'idodi da rashin amfanin su a aikace, kamar su PCR-SSCP, chromatography mai saurin yin ruwa, hanyar binciken binciken oligonucleotide mai kyalli, Hanyar PCR da kuma hadewar ARMS, COLD-PCR hanya, da dai sauransu. Babban aikin chromatography na ruwa yana da ƙayyadaddun bayanai, amma buƙatar samfuran babba ne; PCR-SSCP bashi da tsada da tsada, amma aikin yana da rikitarwa; fasahar gano maye gurbi wanda ya danganci PCR mai kyalli yana da ƙayyadaddun bayanai, ƙwarewa mai girma, da daidaitaccen adadi, Aiki mai sauƙi, cikakken katange amsa da sauran fa'idodi, amma duk suna buƙatar tsara abubuwan share fage da bincike bisa ga nau'in maye gurbi da aka sani, don haka takamaiman shafuka na iya zama gano, kuma duk yiwuwar maye gurbi ba za'a iya gano shi ba.
3. Summary
A takaice, saboda wuraren maye gurbi da hanyoyin ganowa a dakunan gwaje-gwaje daban-daban ba iri daya bane, girman samfuran kwayoyin cutar da aka yi nazari da kuma ingancin hakar DNA suma ba daidai bane, wanda hakan ya haifar da samuwar babban ko karamar sakamakon gwaji tsakanin dakunan gwaje-gwaje Bambancin, daidaitaccen tsari game da gano maye gurbi na KRAS ya zama batun gano asibiti na damuwa a kasashe daban-daban. A halin yanzu, akwai hanyoyi da yawa don gano maye gurbi a cikin kwayar halittar KRAS. Hankali daga sama zuwa ƙasa shine ARMS, pyrosequencing, HRM, ainihin lokacin adadi na PCR, da kuma jerawa kai tsaye. Daga gaskiyar asibiti, ƙarancin hankali ba shi da amfani ga magani na asibiti, amma mahimman hanyoyi masu mahimmanci zasu haifar da ƙayyadaddun ganowa don raguwa, kuma sakamako mara kyau mara ƙima zai iya faruwa kuma ya shafi tsarin shan magani na mai haƙuri. Yin la'akari da abubuwan da ke sama, haɗe tare da hanyar da FDA ta yarda da ita, ana ba da shawarar hanyar ARMS. Tabbas, daga mahangar kasuwa, binciken kwayoyin bai kamata ya jaddada ni ba
thods, amma mayar da hankali kan sakamako daidai na ƙarshe. Dakunan gwaje-gwaje daban-daban na iya ɗaukar hanyoyin gwaji masu dacewa bisa ga ainihin halin da ake ciki, amma kawai idan suna da ingantattun cancantar aiki da tsarin kula da ingancin ciki. A karkashin yanayin muhalli na dakin gwaje-gwaje na gida na yanzu, ya zama dole a gudanar da gwaji a daidaitaccen dakin gwaje-gwaje na PCR da shiga cikin ayyukan kula da ingancin ɗaki na cikin gida da na duniya don tabbatar da ingantaccen ingancin gwajin dakin gwaje-gwaje. Daidaitaccen gudanarwa shine yanayin da ake buƙata don tabbatar da sakamako akai-akai. A kasar Sin, akwai bukatar gaggawa don daidaitawa da daidaita gwajin asibiti na kwayar halittar KRAS, da samar da daidaitattun tsarin gwaji bisa ga bukatu daban-daban, kuma ana iya fadada wannan shirin zuwa gano BRAF, PIK23450_3CA, EGFR da sauran kwayoyin halitta don inganta gwajin cututtukan ƙwayoyin cuta na asibiti.
