Droplet digital PCR as a novel dia-gnostic tool.

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 dia-gnostic test with high sensitivity that identifies the occurrence and level of bio-markers 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 patients 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 dia-gnostics 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.

Detection of driver mutations in FFPE samples from patients with verified malignant melanoma.

Malignant melanoma is an oncological disease characterized by etiologic heterogeneity and it has increasing incidence and mortality in the Slovak Republic. While it is treated surgically in combination with chemotherapy, targeted therapy, and immunotherapy, malignant melanomas can ulcerate and are susceptible to infections. These are highly aggressive cancers with metastasis, and recent studies have shown the presence of mutations in RAC1, PPP6C and STK19 genes in melanoma patients. Mutations in these genes are driver mutations; important in oncogenesis and providing selective advantage to tumor cells. The aim of our study is to establish a method to detect driver mutations in formalin-fixed, paraffin embedded (FFPE) tissue DNA. We applied Sanger sequencing to detect driver somatic mutations in RAC1, PPP6C, STK19 and BRAF genes in patients with malignant melanoma. Confirmation of BRAF V600E mutation was obtained by allele-specific PCR. The BRAF V600E mutation was present in 15 of 113 patients (13.2%) and the driver mutation in 7 of 113 patients (6.2 %). Our results demonstrate that Sanger sequencing analysis detects mutations in FFPE clinical samples. The identification of these somatic driver mutations in samples with verified malignant melanomas enabled development of a molecular classification of melanomas, and our study provides evidence of diversity of novel driver mutations implicated in malignant melanoma pathogenesis. These findings could have very important implications for targeted therapy.

Malignant tumors of the uterine corpus: molecular background of their origin.

Tumors of the uterine corpus can be divided into two main groups: endometrial tumors and mesenchymal tumors. The former ones are common gynecological diseases, whereas malignant mesenchymal tumors, which behave in a much more aggressive way, are quite rare with a poorer prognosis. The most common type of endometrial tumors is endometrioid adenocarcinomas, and in case of mesenchymal tumors, these are carcinosarcomas, or leiomyosarcomas, if only clear types of tumors are taken into account. The objective of this article is to review molecular-genetic abnormalities associated with tumorigenesis of both types of tumors, with focus on the most aggressive forms. This view includes a different expression pattern of genes, usually aberrant in cases of uterine cancer that can arise due to epigenetic modifications, mostly hypermethylation of promoters or microRNA (miRNA)'s interference with concrete genes. Furthermore, clinical predispositions of tumorigenesis, involving hormonal factors, age, and ethnicity, are also mentioned.

[Rapid detection of the most common chromosomal aneuploidies in the second-trimester amniotic fluid using QF-PCR]. / Rýchla detekcia najbeznejsích chromozómových aneuploídií z plodovej vody v druhom trimestri tehotnosti vyuzitím QF-PCR.

OBJECTIVE: To introduce QF-PCR method for detection of the most common chromosomal (trisomy 21, 18 and 13) and gonosomal aneuploidies at our department in the second-trimester amniotic fluid. To test the hypothesis of chromosomal aneuploidies detection using STR markers of Aneufast® kit via analysing free fetal DNA (ffDNA) isolated from plasma of pregnant women with confirmed trisomy 21 in fetus. DESIGN: A prospective clinical study. SETTING: Department of Obstetrics and Gynecology, Jessenius Faculty of Medicine and University Hospital in Martin, Slovak Republic. METHODS: The samples of amniotic fluid were obtained from 67 women (twin pregnancy in 3 cases) in the 2nd trimester (15th to 22nd gestational week (g.w.)). Samples were examined using multiplex QF-PCR via Aneufast kit. In the case of positivity for trisomy 21, they were re-examined using Devyser Resolution 21 kit. All samples were parallelly evaluated by cytogenetic karyotyping. We also analyzed ffDNA from the plasma of 3 high-risk women using Aneufast kit. The plasma samples were obtained in the 2nd trimester(17th to 21st g.w.). Qiaamp DSP Virus kit was used for ffDNA isolation. Trisomy 21 of 3 fetuses was confirmed by karyotyping after 2nd trimester amniocentesis. RESULTS: In the cohort of 70 samples, 7 pathological results (six trisomies 21 and one trisomy 18) were obtained. There was 100% concordance with cytogenetic karyotype in all samples examined by QF-PCR. The amplification of tracked chromosome 21 fragments was not evaluable in the case of ffDNA analysis. CONCLUSION: QF-PCR was approved as reliable, rapid, quite simple and financially bearable method of prenatal diagnostics. Despite the fact of good availability and work implementation of Aneufast® kit, results of ffDNA analysis are insufficient. We did not obtain interpretable results after ffDNA analysis from maternal plasma in trisomy 21 fetuses.

[Our experience with detection of JAK2 mutations in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative disorders]. / Nase skúsenosti s vysetrovaním JAK2 mutácií pacientov s myeloproliferatívnymi ochoreniami z trepanobioptického materiálu kostnej drene.

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by JAK2 mutation. The exon 14 V617F mutation is present in almost all patients with PV and in approx. 60% of patients with ET and PMF. The importance of JAK2V617F in the differential diagnostic considerations is still unclear and here the BM morphology examination still represents an important diagnostic tool. In the WHO classification of Ph1-negative MPNs, the identification of JAK2 mutations represents a major diagnostic criterion of these diseases. Therefore we decided to implement the examination of JAK2V617F mutation in formalin-fixed paraffin-embedded biopsy specimens of patients with Ph1-negative MPN using allele-specific PCR. In addition, in all JAK2 V617F negative patients with PV we sequenced the whole JAK2 exon 12. Until now we examined up to 200 patients with clinically confirmed MPN and our results in all three categories PV, ET and PMF are in agreement with earlier published data. Paraffin embedded tissues represent a valuable source of DNA which can be used in the diagnostics of both JAK2 exon 12 and exon 14 mutations. It is of particular importance if the fresh material is not available and there is a clinical and/or research utility for the performance of PCR on archival bone marrow samples with PV, ET or PMF suspicion.

Hypermethylation of the GSTP1 promoter region in breast cancer is associated with prognostic clinicopathological parameters.

UNLABELLED: Breast cancer is one of the most common cancer affecting women and the recent research is focused on identifying new genetic and epigenetic prognostic and predictive factors. Glutathione S-transferase P1 (GSTP1) is abiotransformation enzyme expressed in normal breast epithelial cells which can be epigenetically inactivated in breast cancer. We have shown, that application of nested two-stage methylation-specific PCR (MSP) is asuitable method for analysis of epigenetically silenced GSTP1 in formalin-fixed paraffin-embedded (FFPE) tissues from breast cancer patients. Of 45 breast tumors, 11 (24, 4%) were found to have methylated GSTP 1promoter region. We were able to demonstrate the correlation between the hypermethylation of the GSTP1 promoter region and histological grade of the tumor (p KEYWORDS: breast cancer, prognostic factors, hypermethylation, GSTP1, methylation-specific PCR.

[Commentary on development of the prognostic factors concept in chronic lymphocytic leukaemia: the route from prognostic factors to therapy response predictors]. / Komentár k vývoju konceptu prognostických faktorov chronickej lymfocytovej leukémie: cesta od prognostických faktorov k prediktorom liecebnej odpovede.

Chronic lymphocytic leukaemia (CLL) is a lymphoproliferative disorder with variable clinical course. Determination of disease prognosis is based on the identification of different prognostic factors. The concept of CLL prognostic factors is still developing and has undergone several fundamental changes. Traditional (old) prognostic factors and staging systems are useful in describing the extent of the disease at any given moment, in determining clinical progression and in the identification of patients who need to start treatment. However, traditional prognostic factors are not sufficient for predicting a long-term prognosis because they are not able to identify potentially aggressive forms of CLL in the early stages. Nevertheless, clinical staging systems maintain their importance and in contrast to other traditional factors also their independent prognostic role. Otherwise, traditional prognostic factors play the role of disease activity descriptors rather than the role of actual prognostic factors. CLL risk profile determination is based on the identification of so-called new prognostic factors, the most relevant of which are chromosomal aberrations, TP53 gene mutations, mutational status of IgVH genes, ZAP-70 and CD38 expression. These factors are able to predict the prognosis already at the time of the initial diagnosis. In contrast to previous ideas, they are not incorporated into recommendations regarding indications for treatment. This is due to the risks associated with early treatment and the lack of data validated in prospective clinical trials demonstrating the justifiability of such procedure. In patients being treated, new prognostic factors may be useful for predicting the response to the therapy and some of them may directly influence the choice of treatment regime. New CLL treatment modalities have also raised the question of their influence on the prognostic and predictive power of new prognostic factors.