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Abstract Background While there is enough convincing evidence in childhood acute lymphoblastic leukemia (ALL), the data on the pre-natal origin in childhood acute myeloid leukemia (AML) are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of non-infant childhood AML and ALL patients for the presence of their respective leukemic markers. Methods We analysed Guthrie cards of 12 ALL patients aged 2–6 years using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements (n = 15) and/or intronic breakpoints of TEL/AML1 fusion gene (n = 3). In AML patients (n = 13, age 1–14 years) PML/RARalpha (n = 4), CBFbeta/MYH11 (n = 3), AML1/ETO (n = 2), MLL/AF6 (n = 1), MLL/AF9 (n = 1) and MLL/AF10 (n = 1) fusion genes and/or internal tandem duplication of FLT3 gene (FLT3/ITD) (n = 2) were used as clonotypic markers. Assay sensitivity determined using serial dilutions of patient DNA into the DNA of a healthy donor allowed us to detect the pre-leukemic clone in Guthrie card providing 1–3 positive cells were present in the neonatal blood spot. Results In 3 patients with ALL (25%) we reproducibly detected their leukemic markers (Ig/TCR n = 2; TEL/AML1 n = 1) in the Guthrie card. We did not find patient-specific molecular markers in any patient with AML. Conclusion In the largest cohort examined so far we used identical approach for the backtracking of non-infant childhood ALL and AML. Our data suggest that either the prenatal origin of AML is less frequent or the load of pre-leukemic cells is significantly lower at birth in AML compared to ALL cases.

Malignant melanoma is a skin tumor arising from melanocytes, occurring mostly in predisposed individuals. Melanomas are frequently present with copy number variations (CNVs), i.e., gains or losses of specific DNA regions that have provided immense potential for disease diagnosis and classification. The methodology of CNV detection has revolutionized in past decades, and current high throughput technologies enable us to analyze the entire spectrum of CNV alterations at the whole genome scale. Thus, identifying novel CNV biomarkers and evaluating their applicability in biomedicine are becoming increasingly important. The aim of this review was to summarize copy number changes occurring in malignant melanomas. We made an overview of specific genes and chromosomal locations affected in sporadic and familial melanoma and also of known germline alterations in melanoma-prone families. We summarized genomic regions aberrant in malignant melanoma and highlighted those frequently discussed in the literature, suggesting 7q, 11q, 12q, 9p, and 1q, but also others, as the most affected ones.

Sticky platelet syndrome (SPS) is a thrombophilia caused by the increased aggregability of platelets in response to the addition of low concentrations of epinephrine (EPI) and/or adenosine diphosphate (ADP). Some of the single nucleotide polymorphisms (SNP), alleles and haplotypes of platelet glycoprotein receptors were proved to have a role in the etiology of thrombotic episodes When comparing SPS and the control group, in VEGFA rs3025039, the p value for both CC vs. TT and CT vs. TT analyses was <0.001. Interestingly, no minor TT genotype was present in the SPS group, suggesting the thrombotic pathogenesis of recurrent spontaneous abortions (RSA) in these patients. Moreover, we found a significant difference in the presence of AT containing a risky A allele and TT genotype of ALPP rs13026692 (p = 0.034) in SPS patients when compared with the controls. Additionally, we detected a decreased frequency of the GG (CC) genotype of FOXP3 rs3761548 in patients with SPS and RSA when compared with the control group (p value for the CC (GG) vs. AA (TT) 0.021). This might indicate an evolutionary protective mechanism of the A (T) allele in the SPS group against thrombotic complications in pregnancy. These results can be used for antithrombotic management in such pregnant patients.

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.

Colorectal cancer (CRC) is a multifactorial disease and one of the most malignant tumours. In addition to the sporadic form, familial occurrences, particularly hereditary non-polyposis CRC-Lynch syndrome (LS)-are often observed. LS is caused by a germline mutation in mismatch repair (MMR) genes, whose task it is to correct errors in the DNA structure that result from its replication. The aim of the present study was to stratify CRC patients using molecular diagnostics and next generation sequencing, according to the chosen criteria [positive for microsatellite instability (MSI) and negative for a BRAF mutation and MutL homolog 1 ( MLH1 ) methylation], and subsequently to detect pathological germline mutations in MMR genes in Slovak patients. To exclude patients with MSI from further testing, the present study detected the BRAF V600E mutation and examined MLH1 methylation status. From the 300 CRC patients, 37 cases with MSI were identified. In the MSI-positive samples, 13 cases of BRAF V600E mutation were recorded. In 24 BRAF -negative patients, 11 cases of epigenetic methylation of MLH1 and 12 cases without MLH1 methylation suspected for LS were detected, and it was not possible to analyse the methylation phenotype of 1 sample. Thus, the present study reports the novel deletion of four nucleotides, 1627&#95;1630del AAAG (Glu544Lysfs*26) in MSH6 , probably associated with LS. A second case with a nonsense mutation in MSH was also detected, namely MMR&#95;c.1030C>T (p.Q344X).

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.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of gastrointestinal tract and are characterized by presence of mutations in tyrosine kinases cKIT (KIT) and PDGFRα (PDGFRA). Mutations identified are highly heterogeneous, but some mutations are associated with specific clinical features of the tumor. Samples from 278 GIST patients collected during the period 2004-2011 were screened for mutations in exons 9, 11, 13, and 17 of KIT and 12, 14 and 18 of PDGFRA. Results of mutation screening were summarized and tested for possible association with clinical parameters of tumors. Mutations were identified in 83.81% of patients. Most frequent mutations were found in KIT exon 11 reaching frequency of 62.95%. Other exons contributed to the mutation pool with frequencies 8.27%, 7.55%, 2.52%, 1.44%, 1.08%, and 0.00%, in decreasing order KIT exon 9, PDGRFA exons 18 and 12, KIT exon 13, PDGFRA exon 14, and KIT exon 17. General linear model analysis showed no effect of any individual analyzed mutation on the phenotypic variables, but we confirmed association between mutations KIT exon 9 p. 503-504&#95;dup2, and PDGFRA exon 18 p. D842V and intestinal and gastric localization of tumors., (© 2012 APMIS Published by John Wiley & Sons Ltd.)

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.

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.