Novel TG-FGFR1 and TRIM33-NTRK1 transcript fusions in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is most common among all thyroid cancers. Multiple genomic alterations occur in PTC, and gene rearrangements are one of them. Here we screened 14 tumors for novel fusion transcripts by RNA-Seq. Two samples harboring RET/PTC1 and RET/PTC3 rearrangements were positive controls whereas the remaining ones were negative regarding the common PTC alterations. We used Sanger sequencing to validate potential fusions. We detected 2 novel potentially oncogenic transcript fusions: TG-FGFR1 and TRIM33-NTRK1. We detected 4 novel fusion transcripts of unknown significance accompanying the TRIM33-NTRK1 fusion: ZSWIM5-TP53BP2, TAF4B-WDR1, ABI2-MTA3, and ARID1B-PSMA1. Apart from confirming the presence of RET/PTC1 and RET/PTC3 in positive control samples, we also detected known oncogenic fusion transcripts in remaining samples: TFG-NTRK1, ETV6-NTRK3, MKRN1-BRAF, EML4-ALK, and novel isoform of CCDC6-RET.

Coexistence of TERT Promoter Mutations and the BRAF V600E Alteration and Its Impact on Histopathological Features of Papillary Thyroid Carcinoma in a Selected Series of Polish Patients.

TERT promoter (TERTp) mutations are important factors in papillary thyroid carcinomas (PTCs). They are associated with tumor aggressiveness, recurrence, and disease-specific mortality and their use in risk stratification of PTC patients has been proposed. In this study we investigated the prevalence of TERTp mutations in a cohort of Polish patients with PTCs and the association of these mutations with histopathological factors, particularly in coexistence with the BRAF V600E mutation. A total of 189 consecutive PTC specimens with known BRAF mutational status were evaluated. TERTp mutations were detected in 8.5% of cases (16/189) with the C228T mutation being the most frequent. In six of the PTC specimens (3.2%), four additional TERTp alterations were found, which included one known polymorphism (rs2735943) and three previously unreported alterations. The association analysis revealed that the TERTp hotspot mutations were highly correlated with the presence of the BRAF V600E mutation and their coexistence was significantly associated with gender, advanced patient age, advanced disease stage, presence of lymph node metastases, larger tumor size, and tumor-capsule infiltration. While correlations were identified, the possibility of TERTp mutations being key molecular modulators responsible for PTC aggressiveness requires further studies.

Age at diagnosis and gender modify the risk of 9q22 and 14q13 polymorphisms for papillary thyroid carcinoma.

INTRODUCTION: Papillary thyroid cancer (PTC) shows familial occurrence, and some susceptibility single nucleotide polymorphisms (SNPs) have been identified in FOXE1 and near the NKX2-1 locus. The aim of our study was to analyse the association of PTC risk with SNPs in FOXE1 (rs965513, rs1867277, rs1443434) and near the NKX2-1 locus (rs944289) in a Polish population, and, in the second step, the interac-tion between SNPs and patient-related factors (age at diagnosis and gender). MATERIAL AND METHODS: A total of 2243 DNA samples from PTC patients and 1160 controls were included in the study. The SNP analysis was performed with the allelic discrimination technique. RESULTS: There were significant associations of all SNPs with PTC (rs965513 odds ratio [OR] = 1.72, p = 8 × 10-7; rs1867277 OR = 1.59, p = 1 × 10-6; rs1443434 OR = 1.53, p = 1 × 10-5; rs944289 OR = 1.52, p = 4 × 10-5). Logistic regression analysis revealed an increased PTC risk in the interaction of rs944289 with age at diagnosis (OR = 1.01 per year, p = 6 × 10-4) and a decreased PTC risk in the interaction of male gender with the GGT FOXE1 protective haplotype (OR = 0.69, p = 0.01). CONCLUSIONS: the association between PTC and all analysed SNPs was confirmed. It was also shown that patient-related factors modify the predisposition to PTC by increasing the risk for rs944289 per year of age, and by enhancing the protective effect of the FOXE1 GGT haplotype in men.

Brain Metastasis Prediction by Transcriptomic Profiling in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) lacks expression of steroid hormone receptors (estrogen receptor α and progesterone) and epidermal growth factor receptor type 2. This phenotype shows high metastatic potential, with particular predilection to lungs and brain. Determination of TNBC transcriptomic profiles associated with high risk of brain metastasis (BM) might identify patients requiring alternative, more aggressive, or specific preventive and therapeutic approaches. PATIENTS AND METHODS: Using a cDNA-mediated annealing, selection, extension, and ligation assay, we investigated expression of 29,369 gene transcripts in primary TNBC tumor samples from 119 patients-71 in discovery cohort A and 48 in independent cohort B-that included best discriminating genes. Expression of mRNA was correlated with the occurrence of symptomatic BM. RESULTS: In cohort A, the difference at the noncorrected P < .005 was found for 64 transcripts (P = .23 for global test), but none showed significant difference at a preset level of false-discovery rate of < 10%. Of the 30 transcripts with the largest differences between patients with and without BM in cohort A, none was significantly associated with BM in cohort B. CONCLUSION: Analysis based on the primary tumor gene transcripts alone is unlikely to predict BM development in advanced TNBC. Despite its negative findings, the study adds to the knowledge on the biology of TNBC and paves the way for future projects using more advanced molecular assays.

The frequency of polymorphic variants of filaggrin gene and clinical atopic dermatitis.

INTRODUCTION: As far as pathogenesis of the atopic dermatitis (AD) is concerned, the roles of an impaired epidermal barrier and cornified cell envelope are widely emphasized. AIM: The assessment of mutations of the filaggrin gene and their connection with the clinical picture of AD as well as selected allergological and environmental indicators. MATERIAL AND METHODS: 105 patients with diagnosed AD on the basis of diagnostic criteria were included. For every patient of the examined group, quantitative determination of the total concentration of IgE and the concentration of IgE antibodies to selected allergens were examined. For all patients, studies were performed by means of analysis of two genomic gene variants of profilaggrin (FLG) - R501X and 2282del4. RESULTS: Loss-of-function mutations in the filaggrin gene were shown in 12 (11.4%) patients in the examined group. All patients in the study group who developed one of the tested loss-of-function mutations in the filaggrin gene demonstrated an extrinsic, allergic form of atopic dermatitis. A significant association (p = 0.0002) between the presence of one of the tested loss-of-function mutations in the filaggrin gene and elevated levels of total concentration of immunoglobulin E was shown. CONCLUSIONS: Patients with AD of null mutations in the filaggrin gene demonstrate a relationship with the total and specific concentration of immunoglobulin E, specifically higher concentrations of IgE against aeroallergens and alimentary allergens as well as elevated levels of total immunoglobulin E.

Molecular classification of pituitary adenomas: in search for criteria useful for high-throughput studies.

INTRODUCTION: The mechanism of pathogenesis of pituitary adenomas is still unknown, and it shows differences in pituitary cells of different origin. The aim of our study was to analyse the gene expression profile of pituitary hormones and their precursor genes: PRL, GH, POMC, TSHb, LHb, FSHb, and CGA by QPCR in particular types of pituitary adenomas, and to evaluate the results in the context of sample selection for microarray studies. MATERIAL AND METHODS: Analysis of the gene expression profile was performed in 84 samples of pituitary adenomas, by real-time quantitative PCR (QPCR). RESULTS: As expected, expression of GH gene was significantly higher in somatotropinomas than in prolactinomas (p < 0.05). For POMC gene we noticed lower expression in all pituitary adenomas, except adrenocorticotropinomas (p < 0.05). In the case of PRL gene, the highest expression was observed; PRL+ adenomas were in third place. LHb and FSHb genes showed the highest expression, respectively, in LH-producing and FSH-producing pituitary adenomas; however, our analysis did not show statistically significant differences between LH-producing and FSH-producing adenomas. CONCLUSIONS: Our study showed that GH is a characteristic gene for somatotropinomas. We drew a similar conclusion for POMC gene and adrenocorticotropinomas. However, the results that we obtained for PRL, TSHb, LHb, FSHb, and CGA genes indicate that evaluation of gene expression is not sufficient for classification of particular subtypes of pituitary adenomas.

Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies.

BACKGROUND: Infinium HumanMethylation 450 BeadChip Arrays by Illumina (Illumina HM450K) are among the most popular CpG microarray platforms widely used in biological and medical research. Several recent studies highlighted the potentially confounding impact of the genomic variation on the results of methylation studies performed using Illumina's Infinium methylation probes. However, the complexity of SNPs impact on the methylation level measurements (ß values) has not been comprehensively described. RESULTS: In our comparative study of European and Asian populations performed using Illumina HM450K, we found that the majority of Infinium probes, which differentiated two examined groups, had SNPs in their target sequence. Characteristic tri-modal or bi-modal patterns of ß values distribution among individual samples were observed for CpGs with SNPs in the first and second position, respectively. To better understand how SNPs affect methylation readouts, we investigated their impact in the context of SNP position and type, and of the Illumina probe type (Infinium I or II). CONCLUSIONS: Our study clearly demonstrates that SNP variation existing in the genome, if not accounted for, may lead to false interpretation of the methylation signal differences suggested by some of the Illumina Infinium probes. In addition, it provides important practical clues for discriminating between differences due to the methylation status and to the genomic polymorphisms, based on the inspection of methylation readouts in individual samples. This approach is of special importance when Illumina Infinium assay is used for any comparative population studies, whether related to cancer, disease, ethnicity where SNP frequencies differentiate the studied groups.

The Risk of Relapse in Papillary Thyroid Cancer (PTC) in the Context of BRAFV600E Mutation Status and Other Prognostic Factors.

INTRODUCTION: The risk of over-treatment in low-advanced PTC stages has prompted clinicians to search for new reliable prognostic factors. The presence of BRAF mutation, the most frequent molecular event in PTC, seems to be a good candidate. However, there is still lack of randomised trials and its significance has been proved by retrospective analyses, involving a large group of patients. The question arises whether this factor is useful in smaller populations, characterised for specialised centres. Thus, the aim of the study was to evaluate the use of BRAF mutation as a potential predictive marker in PTC patients. MATERIAL: 233 PTC subjects treated between 2004-2006, were retrospectively analysed. Stage pT1 was diagnosed in 64.8% patients and lymph node metastases in 30.9%. Median follow-up was 7.5 years. BRAFV600E mutation was assessed postoperatively in all cases. RESULTS: BRAF V600E mutation was found in 54.5%. It was more frequent in patients > 45 years (p=0.0001), and associated with larger tumour size (p=0.004). Patients with tumours <= 10 mm were over-represented among BRAF negative population (p=0.03). No association between BRAF mutation and other clinicopathological factors was observed. BRAF status was associated neither with relapse nor with disease-free survival (DFS) (p=0.76). Nodal status, extrathyroidal invasion and tumour size significantly influenced DFS. CONCLUSION: The risk of PTC recurrence is mainly related to the presence of lymph node metastases and extrathyroidal invasion, whereas no impact of BRAF V600E mutation has been demonstrated.

The prevalence of somatic RAS mutations in medullary thyroid cancer - a Polish population study.

INTRODUCTION: Somatic RET mutations are detectable in two-thirds of sporadic cases of medullary thyroid cancer (MTC). Recent studies reported a high proportion of RAS somatic mutations in RET negative tumours, which may indicate RAS mutation as a possible alternative genetic event in sporadic MTC tumorigenesis. Thus, the aim of the study was to evaluate the frequency of somatic RAS mutations in sporadic medullary thyroid cancer in the Polish population and to relate the obtained data to the presence of somatic RET mutations. MATERIAL AND METHODS: Somatic mutations (RET, RAS genes) were evaluated in 78 snap-frozen MTC samples (57 sporadic and 21 hereditary) by direct sequencing. Next, three randomly selected RET-negative MTC samples were analysed by the next generation sequencing. RESULTS: RAS mutation was detected in 26.5% of 49 sporadic MTC tumours. None of all the analysed samples showed N-RAS mutation. When only RET-negative samples were considered, the prevalence of RAS mutation was 68.7%, compared to 6% observed in RET-positive samples. Most of these mutations were located in H-RAS codon 61 (72%). None of 21 hereditary MTC samples showed any RAS mutations. CONCLUSIONS: RAS mutations constitute a frequent molecular event in RET-negative sporadic medullary thyroid carcinoma in Polish patients. However, their role in MTC tumorigenesis remains unclear.

Unsupervised analysis of follicular thyroid tumours transcriptome by oligonucleotide microarray gene expression profiling.

INTRODUCTION: Mechanisms driving the invasiveness of follicular thyroid cancer (FTC) are not fully understood. In our study, we undertook an unsupervised analysis of the set of follicular thyroid tumours (adenomas (FTA) and carcinomas) to verify whether the malignant phenotype influences major sources of variability in our dataset. MATERIAL AND METHODS: The core set of samples consisted of 52 tumours (27 FTC, 25 FTA). Total RNA was analysed by oligonucleotide microarray (HG-U133 Plus 2.0). Principal Component Analysis (PCA) was applied as a main method of unsupervised analysis. RESULTS: An analysis of biological character of genes correlated to the first six PCs was performed. When genes correlated to the first PC were used to cluster FTC and FTA, they appeared in two branches; one, relatively enriched in adenomas, with homogenous expression of subset of genes, and the other containing mainly carcinomas, with down-regulation of these genes and heterogeneous up-regulation in a smaller cluster of transcripts. Genes highly up-regulated in adenomas included some thyroid-specific transcripts. The second cluster of genes, up-regulated in carcinomas, contained mainly immunity-related transcripts. Immune response genes were found in the first, third and sixth principal components, improving the discrimination between carcinomas and adenomas. CONCLUSIONS: Our unsupervised analysis indicates that invasiveness of follicular tumours might be considered as the major source of variability in transcriptome analysis. However, the distance between both groups is small and the clusters are overlapping, thus, unsupervised analysis is not sufficient to properly classify them.

Current genetic methodologies in the identification of disaster victims and in forensic analysis.

This review presents the basic problems and currently available molecular techniques used for genetic profiling in disaster victim identification (DVI). The environmental conditions of a mass disaster often result in severe fragmentation, decomposition and intermixing of the remains of victims. In such cases, traditional identification based on the anthropological and physical characteristics of the victims is frequently inconclusive. This is the reason why DNA profiling became the gold standard for victim identification in mass-casualty incidents (MCIs) or any forensic cases where human remains are highly fragmented and/or degraded beyond recognition. The review provides general information about the sources of genetic material for DNA profiling, the genetic markers routinely used during genetic profiling (STR markers, mtDNA and single-nucleotide polymorphisms [SNP]) and the basic statistical approaches used in DNA-based disaster victim identification. Automated technological platforms that allow the simultaneous analysis of a multitude of genetic markers used in genetic identification (oligonucleotide microarray techniques and next-generation sequencing) are also presented. Forensic and population databases containing information on human variability, routinely used for statistical analyses, are discussed. The final part of this review is focused on recent developments, which offer particularly promising tools for forensic applications (mRNA analysis, transcriptome variation in individuals/populations and genetic profiling of specific cells separated from mixtures).

The effect of pimecrolimus on expression of genes associated with skin barrier dysfunction in atopic dermatitis skin lesions.

The mechanism of action of pimecrolimus (PIM) on atopic lesions is still under consideration. Thus far, we have evidence of its anti-inflammatory and immunomodulatory activity, and recent papers focus on its effect on epidermal barrier function. This study analysed changes in the expression of genes associated with skin barrier dysfunction in atopic dermatitis (AD) skin lesions after 2 weeks of exposure to PIM 1% cream. A real-time quantitative PCR analysis of selected epidermal differentiation complex genes and three alternative pathway keratins was performed in skin biopsies from 11 individuals with AD before and after PIM exposure. The real-time quantitative PCR analysis was compared to non-lesional skin in the same patients. Involucrin, a small proline-rich region (SPRR) 2C gene, and alternative pathway keratin 16 showed significant over-expression in lesional skin followed by significant decrease after PIM therapy. The SPRR1A gene, S100A9, and keratin 6A were also increased; however, the decrease after PIM treatment was not significant. The changes in S100 A2, A7 and A8 followed a similar course with borderline significance. SPRR4 had a significant decrease in expression in lesional versus non-lesional skin, which persisted after PIM treatment. No significant changes were detected in mRNA expression levels of filaggrin and loricrin. Our results suggest that PIM can be effective in restoring the epidermal barrier in patients with AD at least in part by its impact on expression of genes, which are important for the normal barrier function of skin.