Východiská: Podstatou moderných postupov liečby onkologických pacientov je v dnešnej dobe zacielenie konkrétnych molekúl zapojených do bunkovej signalizácie asociovanej s nádorovou iniciáciou a progresiou. Úspech uvedeného prístupu závisí od správne zvoleného diagnostického testu s vysokou citlivosťou, ktorý identifikuje výskyt a hladinu vybraných biomarkerov u pacientov pre selekciu tých, ktorí budú na liečivo reagovať a budú z neho benefitovať. Vývoj nových technológií a modernizácia tých známych, prispievajú k inováciám molekulárnej charakterizácie karcinómov, ktorá umožňuje detekciu mutačného stavu pacienta s vysokou citlivosťou a špecifickosťou. Cieľ: V práci diskutujeme o využití polymerázovej reťazovej reakcie (PCR) tretej generácie, tzv. droplet digitálnej PCR (ddPCR), v molekulárnej diagnostike karcinómov. Podľa štúdií uvedených v našom prehľade predstavuje ddPCR sľubný nástroj pri vytváraní genetického profilu pacientov s onkologickým ochorením. Optimalizácia a presná validácia môžu preto umožniť postupnú implementáciu ddPCR do klinickej praxe v oblasti onkológie.
Background: Nowadays, modern treatment methods for cancer patients are based on targeting specific molecules involved in cellular signaling system associated with tumor initiation and progression. The success of such approach depends on a correctly chosen diagnostic test with high sensitivity that identifies the occurrence and level of biomarkers in patients to select those who will respond and benefit from the treatment. The development of new technologies and the upgrades of the known ones contribute to the innovations in molecular characterization of cancer, which allows the detection of patient’s mutational status with high sensitivity and specificity. Purpose: Here, we discuss the utilization of the third-generation type of polymerase chain reaction (PCR), droplet digital PCR (ddPCR), in the molecular diagnostics of oncology diseases. According to the studies reported in our review, ddPCR represents a promising tool in genetic profiling of cancer patients. Therefore, the optimization and precise validation may enable gradual implementation of ddPCR into clinical practice in the field of oncology.
The proto-oncogene KRAS belongs among the most frequently mutated genes in all types of cancer and is also very important oncogene related to colorectal tumors. The detection of mutations in this gene in primary tumor is a predictive biomarker for the anti-EGFR therapy in metastatic CRC (mCRC); however, the patients with wild-type KRAS can also show resistance to the personalized medicine. The droplet-based digital PCR technology has improved the analytical sensitivity of the mutations detection, which led us to the idea about the optimization of this approach for KRAS testing. In this study, we report the application of ddPCR technology in order to analyze the presence of KRAS mutations in primary tumor and matched metastasis in lymph nodes (LNs) from patients with mCRC and address the question, whether the improvement in the detection method can lower the discrepancies of KRAS mutations detection between the primary tumor and regional LNs. Genomic DNA with wtKRAS and commercial DNA with mtKRAS (G12D) were used to set up the ddPCR reaction. Formalin-fixed paraffin-embedded tissues from primary tumor and positive lymph node from 31 patients with mCRC were analyzed using ddPCR and Sanger sequencing. KRAS status of primary tumors was known; however, the mutation status of lymph nodes was not detected previously. From 31 samples of primary tumors, our results corresponded to results from IVD kit in 30 cases. For one patient, ddPCR detected KRAS mutation in comparison with negative result of the IVD kit. In the samples of metastatic infiltrated LNs, ddPCR detected 16 samples as a WT KRAS and 15 lymph nodes showed positivity for KRAS mutation, whereby Sanger sequencing found KRAS mutations in 8 cases only. We also found two cases where genetic conditions of KRAS gene differed between primary tumor and infiltrated lymph node, both "low-grade" adenocarcinoma. Our study approved that ddPCR method is adequate technique with high sensitivity and in the future may be used as a diagnostic tool for evaluation of KRAS mutations, especially in infiltrated LNs of patients with mCRC.
- MeSH
- kolon patologie MeSH
- kolorektální nádory diagnóza genetika patologie MeSH
- lidé MeSH
- lymfatické uzliny patologie MeSH
- molekulární patologie metody MeSH
- mutantní proteiny genetika MeSH
- polymerázová řetězová reakce metody MeSH
- protoonkogenní proteiny p21(ras) genetika MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Papilomavírusy patria medzi vírusy s dvojvláknovou DNA (dsDNA). Sú schopné navodiť tak tvorbu benígnych, ako aj malígnych nádorov. Spojitosť medzi infekciou ľudskými papilomavírusmi (HPV) a rakovinou krčka maternice bola detailne opísaná len nedávno vďaka profesorovi zur Hausenovi. Avšak existujú zástupcovia HPV vírusov, u ktorých nebola dokázaná asociácia s vytváraním malignít. Preto rozoznávame tzv. vysoko-(HR) a nízkorizikové (LR) typy papilómov. V našej práci opisujeme životný cyklus HPV, molekulárne mechanizmy počas onkogenézy a snažíme sa o porovnanie HR HPV a LR HPV.
Papillomaviruses belong to a group of viruses with double-stranded DNA (dsDNA). These viruses are believed to induce benign as well as malignant tumour growth. Thanks to professor zur Hausen, the connection between the infection by human papillomaviruses (HPV) and cervix cancer was described in detail a few years ago. However, there exist certain types of HPV viruses, in which no association with malignancies was ever demonstrated. Hence, we can divide HPV into „high-risk“ (HR) and „low-risk“ (LR) group. Our work describes the life cycle of HPV, molecular mechanisms of oncogenesis and aims to compare HR HPV and LR HPV within these terms. Key words: viral cell transformation – oncogene protein E6 – oncogene protein E7 – E5 protein HPV-16 – „high-risk“ – „low-risk“ The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 28. 6. 2013 Accepted: 14. 7. 2013
- MeSH
- Alphapapillomavirus MeSH
- DNA virů genetika MeSH
- infekce DNA virem * MeSH
- infekce papilomavirem * genetika MeSH
- interferony MeSH
- lidé MeSH
- nádorová transformace buněk MeSH
- nádorový supresorový protein p53 MeSH
- onkogenní proteiny virové * genetika metabolismus MeSH
- Papillomavirus E7 - proteiny MeSH
- retinoblastomový protein MeSH
- Check Tag
- lidé MeSH
