IGHV gene mutational analysis in chronic lymphocytic leukemia patients using next-generation sequencing

Kronična limfocitna levkemija spada med limfoproliferativne novotvorbe, kjer se v periferni krvi bolnika nahaja ⡥ 5 x 109/L klonalnih limfocitov B, ki izražajo na svoji površini označevalce CD5, CD19, CD20 in CD23. Gre za izjemno heterogeno bolezen, pri kateri je način zdravljenja, če je potrebno, odvisen od kliničnih, biokemičnih, citogenetskih ter molekularno genetskih parametrov. Neodvisen napovedni dejavnik poteka bolezni je določitev mutacijskega statusa preurejenega variabilnega dela gena za težko verigo imunoglobulina. Ločimo mutiran gen, ki je z zaporedjem najbližje zarodne imunoglobulinske proge skladen v manj ali enako kot 98%, ter nemutiran gen, ki je skladen v več kot 98%. Zlati standard določanja mutacijskega statusa je pomnoževanje tega gena z verižno reakcijo s polimerazo in sekvenciranje po Sangerju. V magistrski nalogi smo vrednotili ustreznost določanja z metodo sekvenciranja naslednje generacije, ki temelji na sekvenciranju s sintezo. Uporabili smo 25 vzorcev ribonukleinskih kislin, pri katerih je bil mutacijski status določen že z referenčno metodo sekvenciranja po Sangerju, jih analizirali še z novo metodo in primerjali rezultate. Zanimal nas je mutacijski status, delež identičnosti z najbolj skladnim zaporedjem zarodne imunoglobulinske proge, skladnost opredelitve preurejenega variabilnega dela gena in alela za težko verigo imunoglobulina, njegova funkcionalnost, skladnost dobljenih nukleotidnih zaporedij in opredelitev stereotipnega B-celičnega receptorja. Določili smo tudi ponovljivost med serijami pri pripravi in analizi knjižnice treh vzorcev. Pri privzeti meji skladnosti z zarodno imunoglobulinsko progo 98% smo ugotovili 100% ujemanje v mutacijskem statusu med obema metodama. Delež identičnosti se je popolnoma ujemal pri 88% vzorcev, pri odstopajočih primerih je bil z novo metodo vedno višji oz. je bil odstotek mutacij nižji. Podobno smo pri 88% vzorcev določili popolno skladnost genov in alelov. Vsi dobljeni geni so bili funkcionalni in popolnoma skladni v B-celičnem receptorju. Skladnost samega zaporedja nukleotidov je bila v vseh primerih skoraj 100%, pri tem je dolžina dobljenih fragmentov precej variirala in bila vedno daljša kot pri določanju z referenčno metodo. Nova metoda je bila popolnoma ponovljiva in primerljiva z referenčno. Zaradi odlične skladnosti rezultatov, večje občutljivosti, možnosti določitve večih preureditev gena in kvantifikacije, lahko zaključimo, da bi vpeljava metode sekvenciranja naslednje generacije v rutinsko delo veliko doprinesla k obravnavi bolnikov v prihodnosti. Pred tem jo bo potrebno še ustrezno standardizirati in validirati.Chronic lymphocytic leukemia is a common lymphoproliferative neoplasm and is diagnosed by the detection of at least 5000 clonal mature B-lymphocytes per microliter in the peripheral blood that coexpress CD5, CD19, CD20 and CD23. The course of the disease can range from indolent without need for treatment to very aggressive with rapid progress. Treatment depends on clinical presentation, biochemical laboratory parameters, cytogenetic and genetic parameters. The immunoglobulin heavy chain variable region gene mutational status is an established independent prognostic factor in those patients. There is a cutoff of 2% deviation or 98% sequence identity to germline that distinguishes between mutated (⡤98%) and unmutated (>98%) mutational status. Polymerase chain reaction followed by Sanger sequencing is the golden standard for mutational status determination. Our goal was to evaluate the possibility of using next-generation sequencing method for said analysis. We used both methods to analyze RNA samples and compared the results consisting of mutational status, percentage of identity of the rearranged gene and allele to its closest germline, identified rearranged genes and alleles, whether the particular gene rearrangement is productive or unproductive and if it is a member of a major stereotyped B-cell receptor subset. We also tested reproducibility between series of library preparation and analysis of three different samples. We chose the same cutoff value (98%) for percentage of identity to germline when interpreting results using next-generation sequencing. We compared the results with Sanger sequences and found 100% concordance in mutational status determination. 88% of cases were concordant in percentage of identity to the closest germline. In discordant cases sequences obtained using next-generation sequencing were closer to the germline with fewer mutations. Similar there was an identical determination of rearranged genes and alleles in the same number of cases. All genes were functional with corcordance in B-cell receptor subset. Nucleotide sequences obtained by both methods were almost 100% identical with variations in their length, but sequences determined with the new method were always longer. The new method is therefore perfectly repeatable and comparable with the old one. Our results aligned wonderfully, the new method is more sensitive with the possibility of quantification and determination of more than one rearranged gene. We can conclude that introduction of next-generation sequencing method into routine work could improve treatment of patients in the future. Extensive standardisation and validation are of course needed beforehand