Prevalence and Impact of BRAF mutation in patients with concomitant papillary thyroid carcinoma and Hashimoto's thyroiditis: a systematic review with meta-analysis.

Background: Evidence suggests that patients with Hashimoto thyroiditis (HT) are at significantly higher risk of developing papillary thyroid cancer (PTC). However, the course of PTC in patients with both diseases concomitantly has been found to be more indolent than conventional PTC. Additionally, it has been well proven that BRAF mutation results in an aggressive course of PTC. The aims of this meta-analysis were to identify prevalence of BRAF mutation and its impact on clinicopathological features in patients with concomitant PTC-HT. Methods: Medline, Cochrane Library, Scopus, and Web of Science were searched until 16.09.2022, resulting in 227 articles, of which nine studies were included. Summary estimates, comparing patients with (A) BRAF (+) PTC-HT versus BRAF (+) PTC, and (B) BRAF (+) PTC-HT versus BRAF (-) PTC-HT, were generated with Review Manager 5.0. Results: In total, 6395 patients were included in this review. PTC-HT patients had significantly less BRAF mutation than PTC patients (Odds Ratio (OR) (95% Confidence Interval (CI))=0.45 (0.35-0.58), P<0.001). BRAF (+) PTC-HT patients were significantly more likely to have multifocal lesions (OR (95% CI)=1.22 (1.04-1.44), P=0.01) but less likely to have lymph node metastasis (OR (95% CI)=0.65 (0.46-0.91), P=0.01) and extrathyroidal extension (OR (95% CI)=0.55 (0.32-0.96), P=0.03) compared to BRAF (+) PTC patients. BRAF (+) PTC-HT patients were more likely to have multifocal lesions (OR (95% CI)=0.71 (0.53-0.95), P=0.02), lymph node metastasis (OR (95% CI)=0.59 (0.44-0.78), P<0.001) and extrathyroidal extension (OR (95% CI)=0.72 (0.56-0.92), P=0.01) compared to BRAF (-) PTC-HT patients. Conclusion: This meta-analysis highlights that the lower prevalence of BRAF mutation in patients with PTC-HT than conventional PTC may explain the indolent clinicopathological course in this cohort.

Correlation analysis of cortisol concentration in hair versus concentrations in serum, saliva, and urine.

INTRODUCTION: Cortisol concentration is measured in blood, urine, and saliva samples. It has been recently proven that cortisol could also be detected in hair samples. Cortisol measurements in different samples have their own individual characteristics and clinical utility. We aimed to investigate the correlation between hair cortisol concentration and standard cortisol measurements used in clinical practice. MATERIAL AND METHODS: Fifty adult volunteers with a negative history of endocrine disorders were enrolled in the study. Morning serum cortisol (MSC), evening serum cortisol (ESC), evening free salivary cortisol (EFSC), urine free cortisol (UFC), and hair cortisol concentration (HCC) were analysed in all participants. Eventually, 41 volunteers were included into the study, whose cortisol concentration in the 1 mg overnight dexamethasone suppression test (1 mg ONDST) were < 50 nmol/L, and cortisol levels in serum, saliva, and urine were within reference ranges. Hair cortisol concentration test was performed for 20 mg of hair strands of the proximal 1 cm hair segments. RESULTS: Hair cortisol concentration ranged from 0.3036 to 2.65 nmol/mg, and the average value was 0.8125 ± 0.4834 nmol/mg. No significant correlations were found between HCC and MSC (rho = 0.04419, p = 0.7838), HCC and ESC (rho = -0.2071, p = 0.1938), HCC and EFSC (rho = 0.1005, p = 0.532), or HCC and UFC (rho = 0.1793, p = 0.262). CONCLUSIONS: This work is another step in the discussion on the application of HCC determinations in clinical practice. Our results have showed no correlations between HCC and single point cortisol assessment in blood, saliva, and urine in patients with reference cortisol levels.

Clinical implications of GWAS variants associated with differentiated thyroid cancer.

The genetic risk of differentiated thyroid cancer (DTC) probably consists of multiple low-penetrance, single-nucleotide polymorphisms (SNP). Such markers are difficult to uncover by linkage analysis but can be revealed by association studies. Genome-wide association studies (GWASs) have uncovered 31 SNPs associated with DTC. These markers carry a low to moderate risk for DTC, but their cumulative effect increases with each successive risk allele. These data support the important contribution of low penetrance variants in the pathogenesis of DTC. Contrary to somatic mutations such as BRAFV600E, germline variants can be ascertained prior to surgical treatment. Therefore, we hypothesise that GWAS SNPs might impact the clinical course of DTC and we can benefit from this knowledge in choosing a treatment strategy. Several associations between clinical factors and GWAS markers have been reported so far. The most important are associations between rs966423 and mortality (HR = 1.60, p = 0.038), extrathyroidal extension (ETE) (OR = 1.57, p = 0.019); rs965513 and tumour diameter (slope of regression 0.14, p = 0.025), lymph node metastasis (OR = 1.59, p = 0.030) and ETE (OR = 1.29, p = 0.045); rs944289 and distant metastasis (OR = 0.58, p = 0.042); and rs116909374 and lymph node metastasis (OR = 0.61, p = 0.016). These findings show that GWAS SNPs are not only the ignition factors (together with environmental factors) for malignant transformation of thyrocytes but might also impact the clinical course of DTC. Surprisingly, it is not always the risk allele for DTC that is associated with worse clinical outcome. The second interesting observation is that GWAS SNPs show different associations with DTC clinical features depending on their histological subtypes. These point to the complexity of DTC with putatively different roles of genes at different stages of DTC development. (Endokrynol Pol 2019; 70 (5): 423-429).

Fine mapping of 14q13 reveals novel variants associated with different histological subtypes of papillary thyroid carcinoma.

The first two genome wide association studies (GWAS) of papillary thyroid carcinoma (PTC) detected five variants associated with PTC. Two of them (rs944289 and rs116909374) are located at 14q13 making that locus an important target of research into the genetic predisposition to PTC. We aimed at uncovering other variants at 14q13 associated with PTC independently from the GWAS variants. We performed next generation sequencing of the 14q13 region and analyzed the allele frequencies of single nucleotide polymorphisms (SNPs) in n = 90 PTC cases vs. n = 379 EUR controls from the 1,000 Genome Project. The variants associated with PTC were validated in an Ohio cohort of n = 1,216 PTC cases and n = 1,416 controls. Next, we analyzed the association between SNPs and expression of nearby genes and clinical parameters. We showed that rs368187 was associated with PTC (OR = 1.31, p = 2.20 × 10-6 ). Rs1632250, Rs1863347 and rs1755787 showed association with classical PTC (cPTC) (n = 891; OR = 1.24, 2.22 × 10-3 , OR = 1.31, p = 2.15 × 10-4 and OR = 1.24, p = 2.06 × 10-3 , respectively) while variant rs28397092 showed association with follicular variant (n = 243; OR = 1.51, p = 1.36 × 10-3 ). Rs1863347 was associated with suppression of PTCSC3 in unaffected thyroid tissue (p = 0.026). Rs1632250, rs1863347 and rs1755787 showed association with multifocality (OR = 1.85, p = 0.001, OR = 1.98, p = 0.001 and OR = 1.76, p = 0.003 respectively) and N stage (OR = 1.79, p = 0.014, OR = 1.73, p = 0.023 and OR = 1.81, p = 0.013, respectively) in microPTC (n = 328) while rs368187 was associated with M stage (OR = 0.56, p = 0.034) in cPTC. Our results disclose multiple variants associated with PTC and clinical features in the 14q13 superlocus. We suggest that translational genotype/phenotype studies should take into account not only somatic mutations but also germline variants.

Hair cortisol concentration in a population without hypothalamic-pituitary-adrenal axis disorders.

BACKGROUND: Measuring hair cortisol seems to be a good alternative to laboratory tests used thus far in routine endocrine diagnostics, primarily because it is independent of the circadian rhythm of cortisol. Due to the average hair growth of 1 cm per month, the results are related to the average blood cortisol levels over the previous weeks, months or years (depending on the length of the hair sample). OBJECTIVES: The aim of this study is an attempt to apply hair cortisol concentration (HCC) measurements to clinical endocrine diagnostics, based on reference cortisol concentrations in the blood in a population without disorders of the hypothalamic-pituitary-adrenal axis (HPA). MATERIAL AND METHODS: In the final selection process, 44 patients were enrolled in the study, all with negative interviews regarding disorders of the HPA and with reference levels of cortisol concentration obtained in routine laboratory tests. In the pre-analytic phase, we used 1 cm proximal hair strands cut from the posterior vertex area of the head, followed by the incubation of a 20 mg hair sample in methanol. The final cortisol measurement was done using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of HCC ranged from 2 pg/mg up to 51.63 pg/mg. The diurnal decrease in cortisol levels was significantly lower in females than in males (p = 0.031), but we do not consider that difference to be clinically significant. The difference in the HCC between males and females was not statistically significant (p = 0.767). The linear regression coefficient for age was not statistically significant (p = 0.847). Neither the regression coefficients for gender nor the gender and age interactions were statistically significant (p = 0.815). CONCLUSIONS: Hair cortisol concentration measurement, unlike other endocrinological tests, gives information about the cortisol concentration in the long-term perspective. The results obtained in this study may be used as a reference for further research aimed at determining normal values of HCC.

Papillary Thyroid Carcinoma: Association Between Germline DNA Variant Markers and Clinical Parameters.

BACKGROUND: Papillary thyroid cancer (PTC) is reported to be highly heritable in epidemiological studies. Genome-wide association studies (GWAS) have uncovered several variants associated with PTC predisposition. It remains unknown whether these variants might contribute to better clinical stratification of PTC patients. METHODS: In order to assess the usefulness of germline genetic analyses in the management of PTC patients, the genotypes of five variants (rs965513, rs944289, rs116909374, rs2439302, and rs966423) were determined in 1216 PTC patients and 1416 controls. Additionally, the expression of seven genes located close to GWAS variants (PTCSC3, MBIP, NKX2-1, FOXE1, DIRC3, PTCSC2, and NRG1) were measured in 73 PTC paired tumor/normal tissues, respectively. Next, the association was analyzed between the genotypes of the germline variants and the levels of gene expression with clinical/pathological features such as age, sex, TNM staging, multifocality status, extrathyroidal expansion, and MACIS score. RESULTS: The risk allele of rs965513 was associated with larger tumor size (p = 0.025) and extrathyroidal expansion (odd ratio [OR] = 1.29, p = 0.045). The variant rs2439302 showed association with lymph node metastasis (OR = 1.24, p = 0.016), and multifocality status of the tumor (OR = 1.24, p = 0.012). The expression of MBIP was associated with T stage (p = 0.010). MBIP and PTCSC3 displayed lower expression in PTC tissue in males than in females (p = 0.025 and p = 0.036, respectively). NKX2-1 displayed lower expression in patients with N1 stage (p = 0.040). CONCLUSIONS: The studied germline risk alleles predisposing to PTC were associated with a more aggressive course of the disease reflected by larger tumor diameter, higher multifocality rate, and more advanced N stage at the time of diagnosis. These results show that germline variants not only predispose to PTC but also might impact its clinical course. However, these associations were only moderate, and further large multi-ethnic studies are required to evaluate the usefulness of these germline variants in the clinical stratification of PTC patients.

PTCSC3 Is Involved in Papillary Thyroid Carcinoma Development by Modulating S100A4 Gene Expression.

CONTEXT: We previously showed that a long noncoding RNA gene, PTCSC3, located close to the variant rs944289 that predisposes to papillary thyroid carcinoma (PTC) might target the S100A4 gene. OBJECTIVE: The aim was to investigate the impact of PTCSC3 on S100A4 expression and its role in cancer development. DESIGN: S100A4 abundance was analyzed by quantitative PCR (qPCR) in unaffected and tumor tissue from n = 73 PTC patients. The expression of PTCSC3 and S100A4 was studied in BCPAP and TPC-1 cell lines with forced expression of PTCSC3 by qPCR. Expression of S100A4 target genes (VEGF and MMP-9) was studied in the BCPAP cell line with forced expression of PTCSC3 by qPCR, reverse transcriptase PCR, and Western blot. The impact of PTCSC3 on BCPAP motility and invasiveness was analyzed by the Transwell and Matrigel assays, respectively. SETTING: This was a laboratory-based study using cells from clinical samples and thyroid cancer cell lines. MAIN OUTCOME AND MEASURE: We aimed to find evidence for a link between the expression of PTCSC3 and thyroid carcinogenesis. RESULTS: Expression data from PTC cell lines pinpointed S100A4 as the most significantly downregulated gene in the presence of PTCSC3. S100A4 was upregulated in tumor tissue (P = 9.33 × 10(-7)) while PTCSC3 was strongly downregulated (P = 2.2 × 10(-16)). S100A4 transcription was moderately correlated with PTCSC3 expression in unaffected thyroid tissue (r = 0.429, P = .0001), and strongly in unaffected tissue of patients with the risk allele of rs944289 (r = 0.685, P = 7.88 × 10(-5)). S100A4, VEGF, and MMP-9 were suppressed in the presence of PTCSC3 (P = .0051, P = .0090, and P =.0037, respectively). PTC cells expressing PTCSC3 showed reduction in motility and invasiveness (P = 4.52 × 10(-5) and P = 1.0 × 10(-4), respectively). CONCLUSIONS: PTCSC3 downregulates S100A4, leading to a reduction in cell motility and invasiveness. We propose that PTCSC3 impacts PTC predisposition and carcinogenesis through the S100A4 pathway.

Genetic predisposition to papillary thyroid carcinoma: involvement of FOXE1, TSHR, and a novel lincRNA gene, PTCSC2.

CONTEXT: By genome-wide association studies, the risk allele [A] of SNP rs965513 predisposes strongly to papillary thyroid carcinoma (PTC). It is located in a gene-poor region of 9q22, some 60 kb from the FOXE1 gene. The underlying mechanisms remain to be discovered. OBJECTIVE: Our objective was to identify novel transcripts in the 9q22 locus and correlate gene expression levels with the genotypes of rs965513. DESIGN: We performed 3' and 5' rapid amplification of cDNA ends and RT-PCR to detect novel transcripts. One novel transcript was forcibly expressed in a cell line followed by gene expression array analysis. We genotyped rs965513 from PTC patients and measured gene expression levels by real-time RT-PCR in unaffected thyroid tissue and matched tumor. SETTING: This was a laboratory-based study using cells from clinical tissue samples and a cancer cell line. MAIN OUTCOME MEASURES: We detected previously uncharacterized transcripts and evaluated the gene expression levels and the correlation with the risk allele of rs965513, age, gender, chronic lymphocyte thyroiditis (CLT), and TSH levels. RESULTS: We found a novel long intergenic noncoding RNA gene and named it papillary thyroid cancer susceptibility candidate 2 (PTCSC2). Transcripts of PTCSC2 are down-regulated in PTC tumors. The risk allele [A] of rs965513 was significantly associated with low expression of unspliced PTCSC2, FOXE1, and TSHR in unaffected thyroid tissue. We also observed a significant association of age and CLT with PTCSC2 unspliced transcript levels. The correlation between the rs965513 genotype and the PTCSC2 unspliced transcript levels remained significant after adjusting for age, gender, and CLT. Forced expression of PTCSC2 in the BCPAP cell line affected the expression of a subset of noncoding and coding transcripts with enrichment of genes functionally involved in cell cycle and cancer. CONCLUSIONS: Our data suggest a role for PTCSC2, FOXE1, and TSHR in the predisposition to PTC.

Ultra-rare mutation in long-range enhancer predisposes to thyroid carcinoma with high penetrance.

Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

The polymorphism rs944289 predisposes to papillary thyroid carcinoma through a large intergenic noncoding RNA gene of tumor suppressor type.

A genome-wide association study of papillary thyroid carcinoma (PTC) pinpointed two independent SNPs (rs944289 and rs965513) located in regions containing no annotated genes (14q13.3 and 9q22.33, respectively). Here, we describe a unique, long, intergenic, noncoding RNA gene (lincRNA) named Papillary Thyroid Carcinoma Susceptibility Candidate 3 (PTCSC3) located 3.2 kb downstream of rs944289 at 14q.13.3 and the expression of which is strictly thyroid specific. By quantitative PCR, PTCSC3 expression was strongly down-regulated (P = 2.84 × 10(-14)) in thyroid tumor tissue of 46 PTC patients and the risk allele (T) was associated with the strongest suppression (genotype [TT] (n = 21) vs. [CT] (n = 19), P = 0.004). In adjacent unaffected thyroid tissue, the genotype [TT] was associated with up-regulation of PTCSC3 ([TT] (n = 21) vs. [CT] (n = 19), P = 0.034). The SNP rs944289 was located in a binding site for the CCAAT/enhancer binding proteins (C/EBP) α and ß. The risk allele destroyed the binding site in silico. Both C/EBPα and C/EBPß activated the PTCSC3 promoter in reporter assays (P = 0.0009 and P = 0.0014, respectively) and the risk allele reduced the activation compared with the nonrisk allele (C) (P = 0.026 and P = 0.048, respectively). Restoration of PTCSC3 expression in PTC cell line cells (TPC-1 and BCPAP) inhibited cell growth (P = 0.002 and P = 0.019, respectively) and affected the expression of genes involved in DNA replication, recombination and repair, cellular movement, tumor morphology, and cell death. Our data suggest that SNP rs944289 predisposes to PTC through a previously uncharacterized, long intergenic noncoding RNA gene (PTCSC3) that has the characteristics of a tumor suppressor.

Telomere length and telomerase reverse transcriptase gene copy number in patients with papillary thyroid carcinoma.

CONTEXT: The family risk ratio for papillary thyroid carcinoma (PTC) is among the highest of all cancers. Collectively, familial cases (fPTC) and sporadic cases (sPTC) are not known to show molecular differences. However, one study reported that telomeres were markedly shorter and the telomerase reverse transcriptase (TERT) gene was amplified and up-regulated in germline DNA from patients with fPTC compared with sPTC. OBJECTIVE: The aim of this study was to evaluate telomere length and TERT gene amplification and expression in blood samples of fPTC and sPTC patients in a genetically distinct population from the previous study. DESIGN: In 42 fPTC and 65 sPTC patients, quantitative real-time PCR was employed to measure the relative telomere length (RTL) and TERT gene copy number and RNA level. To validate the results using alternative methods, we further studied a subset of the original cohort consisting of randomly chosen fPTC (n = 10) and sPTC (n = 14) patients and controls (n = 21) by assessing both telomere length by flow fluorescent in situ hybridization and TERT gene expression by quantitative real-time PCR. RESULTS: RTL and TERT gene copy number did not differ between fPTC and sPTC (P = 0.957 and P = 0.998, respectively). The mean RTL and TERT gene expression were not significantly different among the groups of the validation series (P = 0.169 and P = 0.718, respectively). CONCLUSION: Our data show no difference between familial and sporadic PTC with respect to telomere length, TERT copy number, or expression in our cohort. Further investigations in additional cohorts of patients are desirable.

Thyroid hormone receptor beta (THRB) is a major target gene for microRNAs deregulated in papillary thyroid carcinoma (PTC).

CONTEXT: Loss of the thyroid hormone receptor is common in tumors. In mouse models, a truncated THRB gene leads to thyroid cancer. Previously, we observed up-regulation of the expression of eight microRNAs (miRs) in papillary thyroid carcinoma (PTC) tumors. OBJECTIVE: Our objective was to determine whether THRB might be inhibited by miRs up-regulated in PTC. DESIGN: The potential binding of miR to the 3'-untranslated region of THRB was analyzed in silico. Direct inhibition by miRs binding to the cloned 3'-untranslated region of THRB was evaluated using luciferase assays. Inhibition of endogenous THRB and its target genes (DIO1 and APP) was examined in cell lines transfected by pre-miRs. The impact on thyroid hormone response element (TRE) was evaluated in promoter assays. Correlations between the expression of THRB and miRs was evaluated in 13 PTC tumor/normal tissue pairs. RESULTS: THRB contains binding sites for the top seven miRs up-regulated in PTC (P = 0.0000002). Direct interaction with THRB was shown for miR-21 and miR-146a. We observed lower levels of THRB transcripts in cell lines transfected with miR-21, -146a, and -221 (down-regulation of 37-48%; P < 0.0001), but not with miR-181a. THRB protein was suppressed down to 10-28% by each of four miRs. Concomitant expression of DIO1 and APP was affected (down-regulation of 32-66%, P < 0.0034 and up-regulation of 48-57%, P < 0.0002, respectively). All four miRs affected TRE activity in promoter assays. Down-regulation of luciferase occurred after transfection with pTRE-TK-Luc construct and each of four miRs. The analysis of tumor/normal tissue pairs revealed down-regulation of THRB in 11 of 13 pairs (1.3- to 9.1-fold), and up-regulation of miR-21, -146a, -181a, and -221 in almost all pairs. CONCLUSIONS: MiRs up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.

Access to selected diagnostic procedures in the management of heart failure patients in Poland - POLKARD 2005.

BACKGROUND: Polish heart failure surveys from 1999 and 2005 indicated that non-invasive and invasive diagnostic procedures in heart failure patients are underused, mostly due to limited availability. AIM: To assess the access to procedures used for the diagnosis and treatment of heart failure in randomly selected outpatient clinics and hospital wards in Poland. METHODS: The study was undertaken in 2005, as a part of the National Project of Prevention and Treatment of Cardiovascular Diseases - POLKARD. The data on non-interventional and interventional procedures were collected from 400 primary care units, 396 secondary outpatient clinics and 259 hospitals, and included cardiology or internal medicine departments. Additionally, the last five patients with diagnosed heart failures were identified, who visited outpatient clinics or were discharged from the hospitals, and their medical records of diagnostic procedures were analysed. RESULTS: Echocardiography was not available in approximately 10% of hospital wards and 13-37% of outpatient clinics, both primary and secondary. Generally, the waiting time for echocardiography in Poland varied from region to region. A one-month waiting time was declared by more than 50% of secondary outpatient clinics and only 11-18% of primary care units, regardless of the community size. On the first day of hospital admission, echocardiography was performed in approximately 10% of patients of internal medicine wards and up to 36% of patients in cardiology departments. The assessment of B-type natriuretic peptide (BNP) or N-terminal pro-B-type natriuretic peptide (NT-proBNP) was generally performed only in a few hospitals, usually in cardiology departments. In primary care units, it was practically not available. Percutaneous coronary interventions, pacemaker or cardioverter-defibrillator implantations were available in approximately 20% of city hospitals, 30-40% of province hospitals, and 60-70% of clinical wards of medical universities. CONCLUSIONS: These data show limited availability of echocardiography in primary care units. It is necessary to continue actions for better accessibility and frequency of performing interventional procedures in patients with heart failure in Poland.

[A case report of posttraumatic deep vein thrombosis of the lower limbs in a patient with factor V Leiden mutation and the congenital absence of the inferior vena cava]. / Opis przypadku pourazowej zakrzepicy zyl glebokich konczyn dolnych u chorego obciazonego mutacja czynnika V typu Leiden oraz wrodzonym brakiem zyly glównej dolnej.

The congenital absence of the inferior vena cava (AIVC) is a rare vessels' malformation which may predispose to the development of thrombosis. Although AIVC is very rare, its occurrence should be considered in young patients, under 40 years old, with deep vein thrombosis (DVT). We are describing a case of a young male with bilateral deep vein thrombosis, in whom we defined three risk factors for DVT--trauma, factor V Leiden and the absence of the inferior vena cava. It is worth to consider the occurrence of malformation of the inferior vena cava in the young patients with deep vein thrombosis even when the other obvious risk factors are present (trauma, factor V Leiden mutation). The clinical state and the diagnostic process are discussed.