Barbara Jarząb

Multivariate analysis of prognostic factors for differentiated thyroid carcinoma in children

Abstract.At most centres, the standard treatment for differentiated thyroid cancer (DTC) comprises total thyroidectomy, radioiodine treatment and thyroid-stimulating hormone (TSH) suppressive therapy. There is, however, considerable disagreement over the appropriate treatment for DTC in children. Some dispute the use of total thyroidectomy and/or question the routine application of iodine-131 therapy in children. The aim of this study was to perform a retrospective analysis of treatment results and prognostic factors for DTC in children treated at our centre. The study included 109 children with DTC (aged 6–17 years). The primary treatment comprised total thyroidectomy in 81 cases, radioiodine therapy in 85 cases and TSH suppressive therapy with l-thyroxine in all patients. Uni- and multivariate analysis of prognostic factors for disease-free survival was performed using the Cox regression method. The actuarial survival rate was 100%, and the 5- and 10-year actuarial disease-free survival rates were 80% and 61% respectively. Univariate analysis revealed that older age, total thyroidectomy and radioiodine treatment had a positive impact on disease-free survival whereas there were no statistical differences with regard to the child’s sex, histological type of cancer or lymph node status. On multivariate analysis, radical surgery was estimated to be the most significant factor (P=0.007) for disease-free survival, while less than total thyroidectomy increased the relative risk of relapse by a factor of 10. Radioiodine treatment decreased the relative risk of relapse by a factor of 5, but with borderline significance (P=0.07). Permanent postoperative complications were observed in 17% of children: in 11 laryngeal palsy occurred, in six there was hypoparathyroidism, and one suffered from both. It is concluded that total thyroidectomy and radioiodine treatment significantly improve recurrence-free survival in children and should be routinely applied even in young children as the primary treatment of DTC.

Molecular prognostic markers in papillary and follicular thyroid cancer: Current status and future directions.

Gene expression profiling shows that, by gene signature, the difference between BRAF-positive and BRAF-negative PTC is so distinct that BRAF-positive cancer may be regarded as a molecular subtype of papillary thyroid cancer (PTC). Since much enthusiasm surrounds the BRAF-oncogene as a molecular prognostic factor, a central focus of our consideration is to weigh the current arguments for and against applying BRAF mutation status of the tumor in clinical practice. The frequency of BRAF mutation in PTC is high-45% on average, with values over 70-80% in some populations. This will mean that implementing BRAF mutation as a factor of poor prognosis will shift many PTC patients, considered up to now as low risk ones, to the more extensive treatment. We estimate that 31% of all PTC patients and 39% of those diagnosed with stage I-II disease will face the risk of overtreatment if the decision will be based on the BRAF-positivity of their tumors. Also, the risk of undertreatment in the young patients with BRAF-negative tumors is evaluated with 26%. We think that, as of now, the evidence-based support for such consequences is still weak. Thus, there is urgent need to look for genes or gene signatures which will be helpful in the stratification of BRAF-positive tumors to specify these with poor prognosis with higher accuracy, needed for clinical decisions. Considering this, in the review we summarize the present status of knowledge on other prognosis-related gene expression changes in papillary and follicular cancer and relate them to he tumors biology.

Analysis options for high-throughput sequencing in miRNA expression profiling

BackgroundRecently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling.Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression.MethodsWe compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors.ResultsHigh correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers.ConclusionsCompared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.

SRGAP1 Is a Candidate Gene for Papillary Thyroid Carcinoma Susceptibility

BACKGROUND Papillary thyroid carcinoma (PTC) shows high heritability, yet efforts to find predisposing genes have been largely negative. OBJECTIVES The objective of this study was to identify susceptibility genes for PTC. METHODS A genome-wide linkage analysis was performed in 38 families. Targeted association study and screening were performed in 2 large cohorts of PTC patients and controls. Candidate DNA variants were tested in functional studies. RESULTS Linkage analysis and association studies identified the Slit-Robo Rho GTPase activating protein 1 gene (SRGAP1) in the linkage peak as a candidate gene. Two missense variants, Q149H and A275T, localized in the Fes/CIP4 homology domain segregated with the disease in 1 family each. One missense variant, R617C, located in the RhoGAP domain occurred in 1 family. Biochemical assays demonstrated that the ability to inactivate CDC42, a key function of SRGAP1, was severely impaired by the Q149H and R617C variants. CONCLUSIONS Our findings suggest that SRGAP1 is a candidate gene in PTC susceptibility. SRGAP1 is likely a low-penetrant gene, possibly of a modifier type.

Metal-proteinase ADAM12, kinesin 14 and checkpoint suppressor 1 as new molecular markers of laryngeal carcinoma

The assessment of gene expression profile in laryngeal cancer allows implementation of molecular biology methods in diagnostics, as well as in prognosticating the course of disease, thus allowing taking most optimal decisions as regards the method of treatment, scope of surgical procedure, or the necessity of adding complementary radiotherapy. The aim of the project was to analyze the gene expression profile in laryngeal cancer using oligonucleotide microarrays, having in mind searching new molecular markers for that carcinoma. The study comprised a group of 43 patients (38 males and 5 females) suffering from squamous cell laryngeal carcinoma, diagnosed and surgically treated in the years 2005–2007 in the ENT Department of the Silesian Medical University in Katowice, Poland. RNA was isolated from frozen tissue fragments, with the use of columns RNeasy Midi and Mini Kit (Qiagen). For the examination of gene expression profile, oligonucleotide microarrays of high density were used, provided by Affymetrix (U 133 2.0 PLUS) containing over 54,000 probes for over 47,000 transcripts. Four genes previously not examined in that respect in laryngeal carcinoma, occurred to be good markers of the neoplasm. They are: metal-proteinase ADAM12, cyclin-dependent kinase 2–CDK2, kinesin 14–KIF14, suppressor 1 of checkpoint–CHES1. The analysis of gene expression profile allows, in laryngeal carcinoma, to point out to new genes, which in future may become molecular markers of the carcinoma.

Gene Expression Analysis in Ovarian Cancer – Faults and Hints from DNA Microarray Study

The introduction of microarray techniques to cancer research brought great expectations for finding biomarkers that would improve patients’ treatment; however, the results of such studies are poorly reproducible and critical analyses of these methods are rare. In this study, we examined global gene expression in 97 ovarian cancer samples. Also, validation of results by quantitative RT-PCR was performed on 30 additional ovarian cancer samples. We carried out a number of systematic analyses in relation to several defined clinicopathological features. The main goal of our study was to delineate the molecular background of ovarian cancer chemoresistance and find biomarkers suitable for prediction of patients’ prognosis. We found that histological tumor type was the major source of variability in genes expression, except for serous and undifferentiated tumors that showed nearly identical profiles. Analysis of clinical endpoints [tumor response to chemotherapy, overall survival, disease-free survival (DFS)] brought results that were not confirmed by validation either on the same group or on the independent group of patients. CLASP1 was the only gene that was found to be important for DFS in the independent group, whereas in the preceding experiments it showed associations with other clinical endpoints and with BRCA1 gene mutation; thus, it may be worthy of further testing. Our results confirm that histological tumor type may be a strong confounding factor and we conclude that gene expression studies of ovarian carcinomas should be performed on histologically homogeneous groups. Among the reasons of poor reproducibility of statistical results may be the fact that despite relatively large patients’ group, in some analyses one has to compare small and unequal classes of samples. In addition, arbitrarily performed division of samples into classes compared may not always reflect their true biological diversity. And finally, we think that clinical endpoints of the tumor probably depend on subtle changes in many and, possibly, alternative molecular pathways, and such changes may be difficult to demonstrate.

The prognostic value of tumor markers doubling times in medullary thyroid carcinoma - preliminary report.

IntroductionCalcitonin (Ct) and carcinoembrional antigen (CEA) are widely used as tumor markers for the post-operative follow-up of patients with medullary thyroid carcinoma (MTC).In patients with elevated serum Ct and CEA their dynamics can be described by calculating the doubling time (DT) - the time, they need to double the serum concentration. Previous reports concluded that the Ct and CEA DT have prognostic value in MTC patients.Patients and methodsWe retrospectively analyzed data of 70 MTC patients with elevated serum Ct or CEA. In total, doubling times were calculated and the DT of the less favorable marker was used to stratify the patients into the low- and high-risk group with the cut-off value of 2 years. The survival analysis was performed using Cox proportional hazard method.ResultsThe doubling time < = 2 years of the less-favorable marker had significant prognostic impact for recurrence-free survival, HR = 2.61 (1.43-4.71) and overall survival, HR = 8.99 (3.51-23.04).ConclusionsThe calcitonin and carcinembrional antigen doubling times of less than two years are negative prognostic factors for MTC recurrence-free and total survival in patients with persistent or recurrent disease. They may be used as predictive factors for more intensive search of disease localization in asymptomatic hypercalcitoninemia and for therapy choice in symptomatic disease.

Novel Genome-Wide Association Study-Based Candidate Loci for Differentiated Thyroid Cancer Risk

CONTEXT Genome-wide association studies (GWASs) on differentiated thyroid cancer (DTC) have identified robust associations with single nucleotide polymorphisms (SNPs) at 9q22.33 (FOXE1), 14q13.3 (NKX2-1), and 2q35 (DIRC3). Our recently published GWAS suggested additional susceptibility loci specific for the high-incidence Italian population. OBJECTIVE The purpose of this study was to identify novel Italian-specific DTC risk variants based on our GWAS and to test them further in low-incidence populations. DESIGN We investigated 45 SNPs selected from our GWAS first in an Italian population. SNPs that showed suggestive evidence of association were investigated in the Polish and Spanish cohorts. RESULTS The combined analysis of the GWAS and the Italian replication study (2260 case patients and 2218 control subjects) provided strong evidence of association with rs10136427 near BATF (odds ratio [OR] =1.40, P = 4.35 × 10(-7)) and rs7267944 near DHX35 (OR = 1.39, P = 2.13 × 10(-8)). A possible role in DTC susceptibility in the Italian populations was also found for rs13184587 (ARSB) (P = 8.54 × 10(-6)) and rs1220597 (SPATA13) (P = 3.25 × 10(-6)). Only the associations between rs10136427 and rs7267944 and DTC risk were replicated in the Polish and the Spanish populations with little evidence of population heterogeneity (GWAS and all replications combined, OR = 1.30, P = 9.30 × 10(-7) and OR = 1.32, P = 1.34 × 10(-8), respectively). In silico analyses provided new insights into the possible functional consequences of the SNPs that showed the strongest association with DTC. CONCLUSIONS Our findings provide evidence for novel DTC susceptibility variants. Further studies are warranted to identify the specific genetic variants responsible for the observed associations and to functionally validate our in silico predictions.

Diagnostic and therapeutic guidelines for gastro-entero-pancreatic neuroendocrine neoplasms (recommended by the Polish Network of Neuroendocrine Tumours)

An increased interest in gastro-entero-pancreatic neuroendocrine neoplasms (GEP NENs) has recently been observed. These are rare neoplasms and their detection in recent years has improved. Over 50% of GEP NENs are carcinoids, and they are usually found incidentally during surgery in the small intestine and appendix and at diagnosis in distant metastases, mainly to the liver. There is a need for co-operation between specialists in various disciplines of medicine in order to work out the diagnostic and therapeutic guidelines. In this publication, we present general recommendations of the Polish Network of Neuroendocrine Tumours for the management of patients with GEP NENs, developed at the Consensus Conference which took place in Kamień Śląski in April 2013. Members of the guidelines working groups were assigned sections of the 2008 guidance to update. In the subsequent parts of this publication, we present the rules of diagnostic and therapeutic management of: - neuroendocrine neoplasms of the stomach and duodenum (including gastrinoma); - pancreatic neuroendocrine neoplasms; - neuroendocrine neoplasms of the small intestine and the appendix; - colorectal neuroendocrine neoplasms. The proposed recommendations by Polish and foreign experts representing different fields of medicine (endocrinology, gastroenterology, surgery, oncology, nuclear medicine and pathology) will be helpful in the diagnosis and treatment of GEP NENs patients.

Novel genetic variants in differentiated thyroid cancer and assessment of the cumulative risk.

A genome-wide association study (GWAS) performed on a high-incidence Italian population followed by replications on low-incidence cohorts suggested a strong association of differentiated thyroid cancer (DTC) with single nucleotide polymorphisms (SNPs) at 9q22.33, 2q35, 20q11.22-q12 and 14q24.3. Moreover, six additional susceptibility loci were associated with the disease only among Italians. The present study had two aims, first to identify loci involved in DTC risk and then to assess the cumulative effect of the SNPs identified so far in the Italian population. The combined analysis of the previous GWAS and the present Italian study provided evidence of association with rs7935113 (GALNTL4, OR = 1.36, 95%CI 1.20–1.53, p-value = 7.41 × 10−7) and rs1203952 (FOXA2, OR = 1.29, 95%CI 1.16–1.44, p-value = 4.42 × 10−6). Experimental ENCODE and eQTL data suggested that both SNPs may influence the closest genes expression through a differential recruitment of transcription factors. The assessment of the cumulative risk of eleven SNPs showed that DTC risk increases with an increasing number of risk alleles (p-trend = 3.13 × 10−47). Nonetheless, only a small fraction (about 4% on the disease liability scale) of DTC is explained by these SNPs. These data are consistent with a polygenic model of DTC predisposition and highlight the importance of association studies in the discovery of the disease hereditability.