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.

Differences in the transcriptome of medullary thyroid cancer regarding the status and type of RET gene mutations.

Medullary thyroid cancer (MTC) can be caused by germline mutations of the RET proto-oncogene or occurs as a sporadic form. It is well known that RET mutations affecting the cysteine-rich region of the protein (MEN2A-like mutations) are correlated with different phenotypes than those in the kinase domain (MEN2B-like mutations). Our aim was to analyse the whole-gene expression profile of MTC with regard to the type of RET gene mutation and the cancer genetic background (hereditary vs sporadic). We studied 86 MTC samples. We demonstrated that there were no distinct differences in the gene expression profiles of hereditary and sporadic MTCs. This suggests a homogeneous nature of MTC. We also noticed that the site of the RET gene mutation slightly influenced the gene expression profile of MTC. We found a significant association between the localization of RET mutations and the expression of three genes: NNAT (suggested to be a tumour suppressor gene), CDC14B (involved in cell cycle control) and NTRK3 (tyrosine receptor kinase that undergoes rearrangement in papillary thyroid cancer). This study suggests that these genes are significantly deregulated in tumours with MEN2A-like and MEN2B-like mutations; however, further investigations are necessary to demonstrate any clinical impact of these findings.

Ratio of proliferation markers and HSP90 gene expression as a predictor of pathological complete response in breast cancer neoadjuvant chemotherapy.

INTRODUCTION: Prediction of response to preoperative breast cancer chemotherapy may offer a substantial optimization of medical management of this disease. The most efficient prediction would be done a priori, before the start of chemotherapy and based on the biological features of patient and tumor. Numerous markers have been proposed but none of them has been applied as a routine. The role of MKI67 and HSP90 expression has been recently suggested to predict treatment sensitivity in HER2-positive breast cancer. The aim of this study was to validate the utility of proliferation based markers (MKI67 and CDK1) and heat shock proteins (namely HSP90) to predict response to chemotherapy in cohort of breast cancer patients treated preoperatively. MATERIAL AND METHODS: Ninety-three patients with breast cancer, all females, mean age 42.2 years, among them 32% T1-T2 patients, 49% T3 patients and 13% with T4 tumor stage, 27% N0, 42% N1, 16% N2, 15% N3 were subjected to initial chemotherapy. The majority of patients (86%) received anthracycline and taxane chemotherapy. Among the patients there were 9 individuals with metastatic disease (M1) at initial presentation, and 11 patients were not treated surgically after initial chemotherapy (no sufficient disease response). From 82 patients operated on, 20 patients (24%) showed pathological complete response (pCR), while in 62 patients there was no pCR. 42% of patients were hormone-sensitive HER2-negative, 20% hormone-sensitive HER2-positive, 9% only HER-positive and 29% with triple negative breast cancer. Four gene transcripts (MKI67, cyclin-dependent kinase 1 [CDK1], heat shock proteins HSP90AA1 and HSP- 90AB1) were analyzed in total RNA isolated from single core obtained during preoperative core needle biopsy by quantitative real-time PCR with fluorescent probes (Universal Probe Library, Roche). Results were normalized to the panel of reference genes. RESULTS: There were no statistically significant differences in MKI67 and CDK1 expression between pCR and no pCR groups (p = 0.099 and 0.35, respectively), although the median expression of both genes was slightly higher in pCR group. In contrast, both HSP90AA1 and HSP90AB1 transcripts showed decreased expression in pCR group (medians 0.77 and 0.55) when compared to no p CR group (median 0.86 and 0.73), statistically significant for HSP90AA1 (p = 0.031) and of borderline significance for HSP90AB1 (p = 0.054). The most significant predictor of pCR was the ratio of CDK1 transcript to HSP90AA transcript. This ratio was significantly higher in CR group (median 0.99) than in no CR group (median 0.68, p = 0.0023), and showed a potential diagnostic utility (area under receiver operating characteristic [ROC] curve 0.72). CONCLUSIONS: HSP90AA1 and AB1 genes exhibit low expression in breast cancers highly sensitive to chemotherapy and may indicate the patients with higher probability of pathological complete response. The ratio of HSP90AA1 to proliferation-related markers (CDK1 or MKI67) may be even better predictor of pCR chance, with higher expression of proliferation genes and lower stress response in patients sensitive to chemotherapy.

BRAFV600E-Associated Gene Expression Profile: Early Changes in the Transcriptome, Based on a Transgenic Mouse Model of Papillary Thyroid Carcinoma.

BACKGROUND: The molecular mechanisms driving the papillary thyroid carcinoma (PTC) are still poorly understood. The most frequent genetic alteration in PTC is the BRAFV600E mutation--its impact may extend even beyond PTC genomic profile and influence the tumor characteristics and even clinical behavior. METHODS: In order to identify BRAF-dependent signature of early carcinogenesis in PTC, a transgenic mouse model with BRAFV600E-induced PTC was developed. Mice thyroid samples were used in microarray analysis and the data were referred to a human thyroid dataset. RESULTS: Most of BRAF(+) mice developed malignant lesions. Nevertheless, 16% of BRAF(+) mice displayed only benign hyperplastic lesions or apparently asymptomatic thyroids. After comparison of non-malignant BRAF(+) thyroids to BRAF(-) ones, we selected 862 significantly deregulated genes. When the mouse BRAF-dependent signature was transposed to the human HG-U133A microarray, we identified 532 genes, potentially indicating the BRAF signature (representing early changes, not related to developed malignant tumor). Comparing BRAF(+) PTCs to healthy human thyroids, PTCs without BRAF and RET alterations and RET(+), RAS(+) PTCs, 18 of these 532 genes displayed significantly deregulated expression in all subgroups. All 18 genes, among them 7 novel and previously not reported, were validated as BRAFV600E-specific in the dataset of independent PTC samples, made available by The Cancer Genome Atlas Project. CONCLUSION: The study identified 7 BRAF-induced genes that are specific for BRAF V600E-driven PTC and not previously reported as related to BRAF mutation or thyroid carcinoma: MMD, ITPR3, AACS, LAD1, PVRL3, ALDH3B1, and RASA1. The full signature of BRAF-related 532 genes may encompass other BRAF-related important transcripts and require further study.

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.

Epidermal differentiation complex (locus 1q21) gene expression in head and neck cancer and normal mucosa.

Epidermal differentiation complex (EDC) comprises a number of genes associated with human skin diseases including psoriasis, atopic dermatitis and hyperkeratosis. These genes have also been linked to numerous cancers, among them skin, gastric, colorectal, lung, ovarian and renal carcinomas. The involvement of EDC components encoding S100 proteins, small proline-rich proteins (SPRRs) and other genes in the tumorigenesis of head and neck squamous cell cancer (HNSCC) has been previously suggested. The aim of the study was to systematically analyze the expression of EDC components on the transcript level in HNSCC. Tissue specimens from 93 patients with HNC of oral cavity and 87 samples from adjacent or distant grossly normal oral mucosawere analyzed. 48 samples (24 tumor and 24 corresponding surrounding tissue) were hybridized to Affymetrix GeneChip Human 1.0 ST Arrays. For validation by quantitative real-time PCR (QPCR) the total RNA from all180 samples collected in the study was analyzed with Real-Time PCR system and fluorescent amplicon specific-probes. Additional set of samples from 14 patients with laryngeal carcinoma previously obtained by HG-U133 Plus 2.0 microarray was also included in the analyses. The expression of analyzed EDC genes was heterogeneous. Two transcripts (S100A1 and S100A4) were significantly down-regulated in oral cancer when compared to normal mucosa (0.69 and 0.36-fold change, respectively), showing an opposite pattern of expression to the remaining S100 genes. Significant up-regulation in tumors was found for S100A11, S100A7, LCE3D, S100A3 and S100A2 genes. The increased expression of S100A7 was subsequently validated by QPCR, confirming significant differences. The remaining EDC genes, including all encoding SPRR molecules, did not show any differences between oral cancer and normal mucosa. The observed differences were also assessed in the independent set of laryngeal cancer samples, confirming the role of S100A3 and LCE3D transcripts in HNC. In HNC of oral cavity only one family of EDC genes (S100 proteins) showed significant cancer-related differences. A number of other transcripts which showed altered expression in HNC require further validation.

Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with epithelial ovarian cancer.

Killer immunoglobulin-like receptors (KIRs) regulate function of NK cells and subsets of T cells. HLA class I molecules are ligands for inhibitory KIRs while specificity of activating KIRs is mainly unknown. Both KIR and HLA genotypes are highly polymorphic. In this study we analyzed associations of KIR and KIR ligand genes with the incidence and clinical course of epithelial ovarian cancer. DNA of 142 patients was analyzed for KIR genes and 103 samples were typed for HLA class I. Control group consisted of 200 healthy individuals, including 83 women, analyzed separately. The frequency of KIR genes in patients and controls were comparable. HLA-C group 1 (ligand for KIR2DL2/3) was more frequent in patients than in controls (86.4% vs. 67.5%, p=0.002). The frequency of KIR2DS4fl was higher in patients with endometrioid cancer (72.3%) compared with other histological subtypes (36.5%, p=0.004) and controls (29.5%, p=0.0001). KIR and KIR ligand genotype did not influence significantly the clinical course of the disease. We conclude that the genotype of KIR ligands is strongly associated with the incidence of epithelial ovarian cancer while KIR2DS4fl confers susceptibility to endometrioid subtype of the disease.

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.

Molecular differential diagnosis of follicular thyroid carcinoma and adenoma based on gene expression profiling by using formalin-fixed paraffin-embedded tissues.

BACKGROUND: Differential diagnosis between malignant follicular thyroid cancer (FTC) and benign follicular thyroid adenoma (FTA) is a great challenge for even an experienced pathologist and requires special effort. Molecular markers may potentially support a differential diagnosis between FTC and FTA in postoperative specimens. The purpose of this study was to derive molecular support for differential post-operative diagnosis, in the form of a simple multigene mRNA-based classifier that would differentiate between FTC and FTA tissue samples. METHODS: A molecular classifier was created based on a combined analysis of two microarray datasets (using 66 thyroid samples). The performance of the classifier was assessed using an independent dataset comprising 71 formalin-fixed paraffin-embedded (FFPE) samples (31 FTC and 40 FTA), which were analysed by quantitative real-time PCR (qPCR). In addition, three other microarray datasets (62 samples) were used to confirm the utility of the classifier. RESULTS: Five of 8 genes selected from training datasets (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) were amplified by qPCR in FFPE material from an independent sample set. Three other genes did not amplify in FFPE material, probably due to low abundance. All 5 analysed genes were downregulated in FTC compared to FTA. The sensitivity and specificity of the 5-gene classifier tested on the FFPE dataset were 71% and 72%, respectively. CONCLUSIONS: The proposed approach could support histopathological examination: 5-gene classifier may aid in molecular discrimination between FTC and FTA in FFPE material.

Epidermal growth factor receptor gene expression in high grade gliomas.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene amplification and protein expression in malignant gliomas (anaplastic astrocytoma, AA and glioblastoma, GBL) were suggested to be correlated with the degree of malignancy. Large deletions within the EGFR gene occur frequently in glioma patients. The aim of our study was to analyse EGFR gene expression by real-time PCR by three different amplicons located across the gene and relate it to the age of patients and EGFR mutation status. MATERIAL AND METHODS: We analysed EGFR gene expression in 75 patients, median age 58 years (range 28-75), 52% of glioblastomas, 39% of anaplastic astrocytomas and 9% of low grade gliomas. EGFR expression was measured by real-time PCR, three amplicons located at exons 2-3, 13-14, and 17-18 junctions were analysed, gene expression was normalized by 18S RNA expression. EGFRvIII deletion was detected by RT-PCR. RESULTS: EGFR was found to be expressed in 61.8% of brain gliomas, with strongly positive expression in 12.2% of them. We simultaneously analysed by RT-PCR the EGFRvIII status and found the deletion in 21.3% of tumours. In our group EGFRvIII mutation was significantly more frequent in patients older than 50 years of age (48.6%) than in younger patients (23.5%, p < 0.05). When only GBL patients were assessed, none of the patients younger than 50 years of age had EGFRvIII mutation, whereas in the older subgroup they constituted 36.67% of subjects. We observed that younger patients (below 50 yrs) had slightly lower EGFR expression in comparison to older patients, but this difference was not statistically significant. CONCLUSIONS: As nearly 1/3 of high grade gliomas do not demonstrate abnormal gene expression levels, EGFR status should be taken into account in any targeted therapy attempt. The significance of EGFR axis-related differences between young and old glioma patients and their impact on the prognosis warrant further study.

Occurrence of BRAF mutations in a Polish cohort of PTC patients - preliminary results.

INTRODUCTION: Genetic alterations involving the mitogen-activated protein kinase (MAPK) pathway are frequently demonstrated in papillary thyroid cancer (PTC). BRAF(V600E), the most frequent mutation in adult patients, is present in approximately 50% of PTC. Most clinical studies have demonstrated an association of BRAF(V600E) mutation with aggressive clinicopathological characteristics and high tumour recurrence, although the results are controversial. In this study we present the preliminary results of BRAF mutation frequence in a group of 88 Polish patients with papillary thyroid cancer (PTC) and relate it to the outcome all DTC patients operated in 2004 and 2005. BRAF (V600E) mutation was diagnosed in 38 (43%) of cases. MATERIAL AND METHODS: The presence of BRAF mutation was evaluated in 88 PTC tumours. DNA was isolated from tissue parafin blocks, and the mutation V600E was evaluated by sequence analysis with an AbiPrism 377 and 3130 xl genetic analyzer (Life Technologies). Statistical analysis was carried out with the use of SPSS 12 software. The chi² and Kaplan-Meyer survival analysis were performed. RESULTS: From all analyzed clinico-pathological factors, only older age positively correlated with BRAF mutation frequency (p = 0.0017). Lymph node/distant metastases, multifocality, and extra-thyroid extension did not correlate with BRAF status. One cancer related death and two reccurences were observed in the BRAF+ group while one relapse was diagnosed in the BRAF- group. CONCLUSIONS: Although many studies document BRAF mutation as a prognostic factor in PTC our results underline that it is too early to consider it as a routine clinical predictive factor.

Genetic predisposition to papillary thyroid cancer.

Approximately 5% of differentiated thyroid cancers are hereditary. Hereditary non-medullary thyroid cancer may occur as a minor component of familial cancer syndromes (e.g. familial adenomatous polyposis) or as a primary feature (familial non-medullary thyroid cancer [FNMTC]). Among FNMTC, PTC is the most common. Although a hereditary predisposition to non-medullary thyroid cancer is well established, the susceptibility genes are poorly known. Up to now, by linkage analysis using microsatellite markers, several putative loci have been described - 1q21, 6q22, 8p23.1-p22, and 8q24; however, validation studies have been unsuccessful. In the present review we discuss the results of linkage analysis and the most recent results of genome wide association studies (GWAS) with high resolution SNP (single nucleotide polymorphism) arrays.

NBL1 and anillin (ANLN) genes over-expression in pancreatic carcinoma.

The aim of the study was to analyze the gene expression profile of pancreatic cancer to derive novel molecular markers of this malignancy. The snap-frozen or RNA-later preserved samples of 18 pancreatic adenocarcinomas, 5 chronic pancreatitis cases and 6 specimens of grossly normal pancreas were used for microarray analysis by HG-U133 Plus 2.0 oligonucleotide Affymetrix arrays. Validation was carried out by real-time quantitative PCR (Q-PCR) in the set of 66 samples: 31 of pancreatic cancer, 14 of chronic pancreatitis and 21 of macroscopically unchanged pancreas. By Principal Component Analysis of the microarray data we found a very consistent expression pattern of normal samples and a less homogenous one in chronic pancreatitis. By supervised comparison (corrected p-value 0.001) we observed 11094 probesets differentiating between cancer and normal samples, while only seventy six probesets were significant for difference between cancer and chronic pancreatitis. The only gene occurring within the best 10 genes in both comparisons was S100 calcium binding protein P (S100P), already indicated for its utility as pancreatic cancer marker by earlier microarray-based studies. For validation we selected two genes which appeared as valuable candidates for molecular markers of pancreatic cancer: neuroblastoma, suppression of tumorigenicity 1 (NBL1) and anillin (ANLN). By Q-PCR, we confirmed statistically significant differences in these genes with a 9.5 fold-change difference between NBL1 expression in cancer/normal comparison and a relatively modest difference between cancer and pancreatitis. For ANLN even more distinct differences were observed (cancer/normal 19.8-fold, cancer/pancreatitis 4.0-fold). NBL1 and anillin are promising markers for pancreatic carcinoma molecular diagnostics.