Ovarian cancer is the fifth most common cancer in women worldwide. Moreover, there are no reliable minimal invasive tests to secure the diagnosis of malignant pelvic masses. Cell-free, circulating ...microRNAs have the potential as diagnostic biomarkers in cancer. Here, we performed and validated a miRNA panel with the potential to distinguish OC from benign pelvic masses.
The profile of plasma microRNA was determined with a panel of 46 candidates in a discovery group and a validation group, each consisting of 190 pre-surgery plasma samples from age-matched patients with malignant (n = 95) and benign pelvic mass (n = 95), by real time RT-qPCR.
Four up-regulated (miR-200c-3p, miR-221-3p, miR-21-5p, and miR-484) and two down-regulated (miR-195-5p and miR-451a) microRNAs were discovered. From those, miR-200c-3p and miR-221-3p were further confirmed in a validation cohort. A combination of these 2 microRNAs together with CA-125 yielded an overall diagnostic accuracy of AUC = 0.96.
We showed consistent plasma microRNA profiles that provide independent diagnostic information of late stage OC.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The MAPK signalling genes KRAS, NRAS and BRAF and the PIK3CA gene are routinely investigated for mutations in the diagnostic routine of colorectal cancer. Few studies have reported co‐existing ...mutations in these genes with clinical relevance, while some have been previously regarded as mutually exclusive. We set to investigate the frequency and co‐occurrent mutations in these targets, and the occurrence of mismatch repair deficiency (dMMR) in a large cohort of Danish colorectal cancers. 1000 colorectal tumours were sequenced as part of our diagnostic workflow for KRAS, NRAS, BRAF and PIK3CA mutations using next‐generation sequencing (NGS) and analysed by immunohistochemistry (IHC) for loss of the MMR proteins, MLH1, PMS2, MSH2 and MSH6. Co‐existing mutations in 12 patients (1.2%) occurred as multiple mutations in the same gene or spread across several genes (KRAS, NRAS and/or BRAF). The frequency of single mutations in the genes occurred with a frequency similar to previously reported, except for a higher frequency of BRAF mutations (18.0%). We found dMMR in 14.6% of the cases with a majority lacking expression of both MLH1 and PMS2. BRAF mutations were only present in dMMR cases involving MLH1 and/or PMS2. Our findings suggest that co‐existing mutations occur, except for the hotspot BRAF V600E, which is mutually exclusive with KRAS/NRAS mutations. Therefore, instead of single gene alterations from the MAPK signalling, assessing co‐occurrence of mutations within one or more of those genes should also be accounted. This may impact future oncological treatments and should be considered in the diagnostic workflow.
Optimized and reliable DNA/RNA extraction protocols are a vital tool in clinical practice in the context of molecular testing. Here, we present our successful attempt to enhance the quantity of RNA ...isolated from clinical specimens, which we originally found challenging (breast and testis). We compared several purification methods with special focus on two AllPrep system-based protocols (QIAGEN). Our data suggest that addition of proteinase K may markedly increase RNA and, in some cases, also DNA yield. The extraction kit used, AllPrep DNA/RNA/miRNA universal kit, provides RNA amounts comparable with the phenol-chloroform extraction method; however, part of the final yield consisted of small RNAs, visible as a thick band in the bioanalyzer gel-like image (5S peak). The 5S peak, albeit in some cases dominating the bioanalyzer image, plays only a small role in RT-qPCR analysis, and Qubit or NanoDrop measurements can still be used as a reliable estimate of starting amounts of mRNA for downstream analyses. In conclusion, we showed that implementing a protocol containing a step of proteinase K digestion markedly increases RNA yield. The AllPrep DNA/RNA/miRNA Universal Kit can be successfully used for simultaneous extraction of DNA and total RNA, irrespective of the tissue of origin, and does not present inconveniences related to phenol-chloroform extraction.
Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of ...tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.
Potential Targeted Therapies in Ovarian Cancer Sisman, Yagmur; Vestergaard, Lau Kræsing; de Oliveira, Douglas Nogueira Perez ...
Pharmaceuticals,
10/2022, Letnik:
15, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Background: We aimed to identify somatic pathogenic and likely pathogenic mutations using next-generation sequencing (NGS). The mutational findings were held against clinically well-described data to ...identify potential targeted therapies in Danish patients diagnosed with high-grade serous ovarian cancer (HGSC). Methods: We characterized the mutational profile of 128 HGSC patients. Clinical data were obtained from the Danish Gynecological Database and tissue samples were collected through the Danish CancerBiobank. DNA was analyzed using NGS. Results: 47 (37%) patients were platinum-sensitive, 32 (25%) partially platinum-sensitive, 35 (27%) platinum-resistant, and three (2%) platinum-refractory, while 11 (9%) patients did not receive chemotherapy. Overall, 27 (21%) had known druggable targets. Twelve (26%) platinum-sensitive patients had druggable targets for PARP inhibitors: one for tyrosine kinase inhibitors and one for immunotherapy treatment. Eight (25%) partially platinum-sensitive patients had druggable targets: seven were eligible for PARP inhibitors and one was potentially eligible for alpesilib and hormone therapy. Seven (20%) platinum-resistant patients had druggable targets: six (86%) were potentially eligible for PARP inhibitors, one for immunotherapy, and one for erdafitinib. Conclusions: PARP inhibitors are the most frequent potential targeted therapy in HGSC. However, other targeted therapies remain relevant for investigation according to our mutational findings.
Uma parcela significativa das lesões na molécula do DNA é causada por espécies reativas de oxigênio e a sua produção excessiva e/ou o funcionamento deficiente dos sistemas celulares antioxidantes, ...que neutralizam a sua ação, é conhecido como estresse oxidativo. Os danos em células normais são prontamente detectados por um sistema de defesa e, em conseqüência, uma rede intrínseca de sinalizações é ativada, sendo que uma das vias resulta na ativação dos mecanismos de reparo do DNA. O reparo por excisão de bases (BER) parece ser a via preferencial de reparo de bases oxidadas, mas existem outras vias de reparo implicadas na reversão do dano oxidativo. A doença de Alzheimer (DA), uma patologia causada particularmente por danos oxidativos, acomete atualmente cerca de 25 milhões de pessoas no mundo, sendo o risco aumentado a partir dos 65 anos de idade. Com isso, a necessidade da identificação de fatores de risco, além de fatores protetores relacionados à DA, tornou-se de grande importância. Por outro lado, há também a necessidade de estudos em nível molecular, que possam fornecer informações sobre os mecanismos que levam ao desenvolvimento da doença. Nesse sentido, foi realizado no presente trabalho, um estudo de expressão gênica transcricional pelo método de microarranjos de DNA, bem como uma análise por PCR em tempo real para uma série de genes envolvidos na resposta ao dano oxidativo no DNA (percepção de danos e reparo do dano), além de outros genes relacionados à doença. Adicionalmente, foram também avaliadas as quebras na fita dupla de DNA causadas por bases oxidadas, em linfócitos de pacientes de Alzheimer (grau moderado) e indivíduos sadios, usando-se métodos de detecção de bases oxidadas (8-oxoGuanina). Entre os vinte genes analisados pelo método de PCR quantitativa em tempo real, apenas a APOE mostrou-se induzida, enquanto 19 genes (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) foram reprimidos transcricionalmente. Essa repressão verificada para a maior parte dos genes estudados indica que várias vias de sinalização celular ligadas a respostas ao estresse oxidativo, incluindo-se as várias vias de reparo do DNA, podem estar envolvidas na condição DA. Adicionalmente, a análise de expressão gênica por microarranjos de cDNA indicou uma série de 41 genes significativamente modulados (q < 0,06) (dentre eles, NOTCH1, MARK3, PAK, SMC1L1) mas para a maioria destes não há relatos na literatura sobre uma possível relação com DA. Por essa razão, o método de microarranjos de cDNA aponta novas vias que possam estar alteradas em DA, o que constitui uma informação importante. Em conjunto, os dados obtidos no presente estudo fornecem uma contribuição relevante, que futuramente poderão contribuir em termos de intervenção terapêutica.
A great amount of DNA molecule lesions is caused by reactive oxygen species and its synthesis in excess and/or misfunctioning of antioxidant cell systems, which neutralize its effects, is known as oxidative stress. Damage in normal cells is readily detected by a defence system and as consequence, a complex signaling pathway is activated, among them DNA repair mechanisms. The base excision repair (BER) seems to be the primary repair pathway in base oxidative damages, however there are other pathways that are involved in their repair. The Alzheimers disease (AD), a pathology caused particularly by oxidative damages, hits 25 million people worldwide, and its prevalence increases every 5 years beyond age 65. Therefore, there is an emerging need of finding risk factors, as well as protective factors related do AD. By the other hand, it is also necessary molecular studies, which could provide precious information about the mechanisms which lead to the disease development. In the present work, it was made a study about transcriptional gene expression by cDNA microarray, as well as Real Time PCR analysis in a series of genes involved in oxidative DNA damage response (sensing and damage repair), and others associated with the disease. In addition, it were also evaluated DNA strand breaks induced by oxidized bases in lymphocytes from Alzheimers patients (moderate level) and healthy individuals, by oxidized bases (8- oxoguanine) detection methods. Among the twenty genes tested by the quantitative Real Time PCR assay, only APOE was induced, as the remaining 19 (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) were found repressed. This observed inhibition in most of genes studied shows that many cell signaling pathways associated to oxidative stress response, including DNA repair pathways, may be also involved in the AD pathology. Additionally, the gene expression analysis by cDNA microarrays showed transcriptional alterations in 41 genes (q < 0.06) (among them, NOTCH1, MARK3, PAK and SMC1L1), but for most of them, there are no reports in the literature about their possible relationship with AD, what brought us new important information. Together, all the data obtained in the preset study provide a relevant contribution, which, in the future, may help on new therapeutic designs.
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