Dr. sc. Sendi Kuret, mol.biol. Klinički zavod za patologiju, sudsku medicinu i citologiju KBC Split Split,
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1 Dr. sc. Sendi Kuret, mol.biol. Klinički zavod za patologiju, sudsku medicinu i citologiju KBC Split Split,
2 Nastanak tumora je proces koji se sastoji od niza promjena na genotipskoj i na fenotipskoj razini. Razumijevanje nastanka patološkog procesa na razini molekularnih promjena osnova je na kojoj bi se trebalo temeljiti sveobuhvatno znanje o nastanku, ali i liječenju određene bolesti, odnosno stanja. Razvoj molekularne medicine (stanična i molekularna razina) dovodi do otkrića i razjašnjenja mehanizama koji potiču nastajanje i rast tumora.
3 Rezultat razvoja molekularne medicine- novi terapijski postupci, koji se nazivaju ciljana (biološka) terapija, koja je usmjerena prema specifičnim molekularnim biomarkerima (EGFR, KRAS,BRAF). Ciljana (biološka ) terapija vodi prema proizvodnji lijekova koji ciljano djeluju na biomarkere, tzv. pametni lijekovi. Novi dijagnostički postupci i»pametni«lijekovi za liječenje raka omogućavaju personalizirani pristup u liječenju bolesnika.
4 Djeluju znatno selektivnije protiv tumorskih stanica, čime ciljana terapija pruža mogućnost veće učinkovitosti i manje toksičnosti ako se uspoređuje sa standardnom kemoterapijom. - avastin (bevacizumab) -za rak debelog crijeva, dojke, pluća i bubrega - zelboraf (vemurafenib)- za melanom - gleevec (imatinib mesylate)- za liječenje mijeloične leukemije i gastrointestinalnog stromalnog tumora (GIST) - herceptin (trastuzumab) -za rak dojke - iressa (gefinitib) i tarceva (erlotinib) -za rak pluća - erbitux (cetuksimab) -za rak debelog crijeva, glave i vrata
5 Budućnost liječenja raka jest u personaliziranoj terapiji koja bi trebala biti prilagođena svakom pacijentu. Jako je važno uraditi genetske analize prije nego što se krene u terapiju ovim ljekovima da bi se vidjelo kakav će biti odgovor na liječenje Važna je suradnja onkologa, patologa i molekularnih biologa radi postavljanja točne dijagnoze i uključivanja ciljane i prilagođene terapije svakom pojedincu.
6 Genetsko profiliranje raka već se obavlja u brojnim laboratorijima diljem svijeta, a jedina prepreka da to postane praksa je cijena takvih analiza. Ovakav pristup predstavlja odmak od tradicionalnih metoda liječenja kod kojih se koristi jedan lijek za sve, prema liječenju prilagođenom svakom pojedinom pacijentu, pravi lijek za pravog pacijenta u pravo vrijeme. Zahvaljujući pametnim lijekovima, individuliziranoj terapiji i ranom otkrivanju malignih bolesti, u svijetu je preživljenje danas čak pet puta više nego prije tridesetak godina.
7 Od 2010.godine radi se molekularno testiranje kojim se utvrđuje genski status KRAS, NRAS, EGFR i BRAF gena u različitim karcinomima (debelo crijevo, pluća, melanom ).
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9 Uzorci za testiranje mutacija - Uzorci tumorskog tkiva fiksirani u formalinu, uklopljeni u parafin (FFPE) - citološki uzorci
10 Dijagnostički protokol I BIOPSIJA PHD dijagnostika DNA ekstrakcija mol. testiranje Dijagnostički protokol II Citološka punkcija CITOLOGIJA DNA ekstrakcija mol. testiranje
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12 EUROPSKO DRUŠTVO ZA PATOLOGIJU Predlaže dva testa ( za kliničku upotrebu-ce): -TheraScreen kit (7 mutacija) -KRAS LightMix kit (9 mutacija)
13 Test Disease cobas BRAF Mutation Test* Metastatic Melanoma cobas KRAS Mutation Test* Colorectal Cancer cobas EGFR Mutation Test* Non-Small Cell Lung Cancer (NSCLC) cobas PIK3CA Mutation Test* Breast Cancer
14 Brzina - Rezultati ispitivanja dostupani u kratkom vremenu Točnost - Visoka osjetljivost i specifičnost Pouzdanost - Dosljedan tijek rada Automatizacija - Automatiziran PCR, interpretacija rezultata i izvještaj
15 1.Priprema uzoraka (cobas DNA Sample Preparation Kit) 2.Amplifikacija i detekcija cobas Mutation Test
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18 cobas EGFR Mutation Test 1. Mutation Detected: Exon 18 G719X 2. Mutation Detected: Exon 19 Deletion 3. Mutation Detected: Exon 20 T790M 4. Mutation Detected: Exon 20 S768I 5. Mutation Detected: Exon 20 Insertion 6. Mutation Detected: Exon 21 L858R 7. Invalid (sample out of range/control failure) 8. Failed (hardware/software failure)
19 cobas EGFR Mutation Test v2 NEW separate sample preparation kit cfdna Sample Preparation Kit cobas cfdna Sample Preparation Kit UPDATED tests both tissue and blood SAME cobas 4800 PLATFORM v2 cobas EGFR Mutation Test v2 cobas 4800 System, v2.1 cobas DNA Sample Preparation Kit
20 cobas EGFR Test v2 for Use with Tissue & Blood Amplification and Detection Kit 42 MUTATIONS DETECTED AMPLIFICATI ON MMX MMX1 MMX2 MMX3 v.2 NEW REAGENT TARGET EX19Del; S768I; EX28/IC L858R; T790M; EX28/IC L861Q; G719A/C/S; EX20Ins; EX28/IC
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22 Od 1. siječnja do 31. prosinca godine 147 tumora (parafinskih blokova) upućeni su na genetsku molekularnu analizu. U 15 (13,5 %) bolesnika utvrđena je mutacija EGFR, dok u 111 bolesnika nije bilo mutacije EGFR. U 21 bolesnika nakon izolacije nije bilo dovoljno DNK za molekularnu analizu. Rezultati analize po tipu mutacije EGFR prikazani su na slici exon 18 G719X exon 19 del exon 20 ins exon 21 L858R
23 ODREĐIVANJE TERAPIJE NA OSNOVI REZULTATA TESTIRANJA EGFR MUTACIJA NEMA MUTACIJA NEMA PODATAKA O OSJETLJIVOSTI NA INHIBITORE TIROZIN KINAZA (TKI) PRISUTNE MUTACIJE IZVJEŠTAJ O TIPU MUTACIJE I NJENOM ZNAČENJU DELECIJA U EKSONU 19 (bilo koje vrste) L858R G719S/A/C ili bilo koja druga substitucija na toj poziciji INSERCIJE U EKSONU 20 T790M DRUGI TIP MUTACIJA L861Q DUPLE MUTACIJE S7681 3% 90 % 7% DOKAZANA OSJETLJIVOST NA DJELOVANJE INHIBITORA TIROZIN KINAZA (TKI) TRENUTNO NEMA PODATAKA O OSJETLJIVOSTI NA DJELOVANJE INHIBITORA TIROZIN KINAZA (TKI) OGRANIČENI PODACI O OSJETLJIVOSTI NA DJELOVANJE INHIBITORA TIROZIN KINAZA (TKI)
24 Test cobas KRAS Detected Mutations Exon 2 Exon LightMix NRAS Exon 2 Exon 3 Exon Exon 4 KRAS exon
25 lijek se ne primjenjuje K-ras mutacija N-ras mutacija Lijek se primjenjuje
26 The cobas 4800 BRAF V600 Mutation Testreal-time PCR test dizajniran za detekciju V600E (T1799A) mutacije.
27 Izazovi ispitivanja molekularnih biomarkera Mnoge varijable i potencijal za pogreške Platform and results Non-validated platforms, manual analysis and reporting Method Many choices benefits /drawbacks unclear Reagents Multi-source reagents, inconsistent quality Specimen Variable quality and quantity of specimen Laboratory Undefined preanalytical steps contribute to different results
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32 U svakodnevnom radu s oboljelima od karcinoma treba implementirati rutinsko testiranje za poznate biomarkere (EGFR, KRAS, NRAS, BRAF), ali i za nove, koji će biti povezani sa specifičnom terapijom.
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