A Rare Case of Isolated Prostate Recurrence From Testicular Seminoma.

We report the case of a 32-year-old male diagnosed with a left-sided testicular seminoma treated with radical inguinal orchiectomy and staged as pT1bN0M0S0 (rete testis invasion) - stage IA. Adjuvant treatment options were discussed, and active surveillance was chosen. Two years later, he presented with urinary retention alternating with pollakiuria, a feeling of incomplete bladder emptying, dyspareunia, and anejaculation. A rectal examination documented an enlarged, nodular, painful prostate. Blood and urine analyses, including serum tumor markers, were unremarkable. Pelvic magnetic resonance (MR) documented a central, nodular, solid, hypermetabolic, prostatic tumor with a size of 40x50x25 mm, invasion of the right seminal vesicle, right anterolateral wall of the rectum, and postero-inferior bladder wall, and an absent lymph node and visceral disease. A transrectal ultrasound-guided (TRUS) biopsy documented prostatic metastasis of the seminoma. The patient was treated with four cycles of bleomycin, etoposide, and cisplatin (BEP) chemotherapy (ChT) with a complete (clinical, radiologic, metabolic, and pathological) response. After five years of follow-up, he remains asymptomatic without a recurrence of the disease.

Significance of Furin Expression in Thyroid Neoplastic Transformation.

Angiotensin-Converting Enzyme 2 (ACE2), Transmembrane Serine Protease 2 (TMPRSS2), and Furin were known to be key players in the SARS-CoV-2 infection, and the thyroid gland was revealed to be one of the relevant targets of the virus. Regardless of the viral infection, the expression of these molecules in the thyroid gland and their putative role in the neoplastic transformation of the thyrocytes has not been thoroughly explored. In this work, we aimed to characterize the mRNA and protein expression pattern of ACE2, TMPRSS2, and Furin in a series of patients with thyroid lesions. Our main results revealed a significantly decreased expression of ACE2 mRNA in the thyroid neoplasms in comparison to normal adjacent tissue. Furin mRNA was significantly increased in thyroid neoplasms when compared to normal adjacent tissue. In addition, a higher Furin mRNA level in thyroid carcinomas was associated with the presence of lymph node metastasis. Furin mRNA expression revealed a high discriminatory power between adjacent tissue and neoplasms. Protein expression of these molecules did not correlate with mRNA expression. Our study shows the mRNA downregulation of ACE2 and overexpression of Furin in thyroid neoplasms. Further studies are required to clarify if Furin expression can be a potential diagnostic indicator in thyroid neoplasia.

Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.

LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.

Detection of SARS-CoV-2 infection in thyroid follicular cells from a COVID-19 autopsy series.

Objective: To understand whether thyroid cells can be directly infected by the SARS-CoV-2 virus and to establish a putative correlation with the expression of the host entry machinery: ACE-2, TMPRSS2, and furin. Methods: We assessed the presence of SARS-CoV-2 virus at the gene level by RT-PCR, viral RNA transcripts localization by in situ hybridization, and by detecting viral proteins by immunohistochemistry for the nucleocapsid and the spike proteins. Furthermore, we also described the immunoexpression of key host factors for virus entry in the COVID-19 thyroid samples. Results: We performed RT-PCR for SARS-CoV-2 in all autopsy specimens and detected viral genome positivity in 13 of 15 thyroid tissues and in a lung specimen. In 9 of the 14 positive samples, we were also able to confirm SARS-CoV-2 signal by in situ hybridization. Immunohistochemistry for the viral nucleocapsid and spike protein was also positive for ten and nine of the RT-PCR-positive cases, respectively, but revealed a lower sensitivity. We also described, for the first time in a COVID-19 series, the immunohistochemical expression of ACE-2, TMPRSS2, and furin in the thyroid. Conclusions: Our results obtained in thyroid specimens from deceased COVID-19 patients indicate that thyrocytes can be directly infected by SARS-CoV-2 since we detected the presence of SARS-CoV-2 genome in follicular cells. Nevertheless, we did not find a clear correlation between the presence of viral genome and the expression of the host factors for virus entry, namely ACE-2, TMPRSS2, and furin.

Pharmacotherapy used for alcohol and cocaine use disorders in a CAPS-AD of Minas Gerais

Abstract Substance use disorder is one of the major social and public health problems in the world. The present study analyzed the pharmacoepidemiological profile of patients treated at the Psychosocial Treatment Center for Alcohol and Substance Use Disorders (CAPS-AD) for treatment of alcohol use disorders (AUD), cocaine use disorders (CUD) and concomitant alcohol and cocaine use disorders (A-CUD) in the city of Betim-MG. The study used quantitative and descriptive data and was based on the evaluation of medical records of patients attended from January to December 2016. After analyzing 295 medical records, the majority of study participants were male (83.7 %) with an average age of 46.26 for AUD, 28.88 for CUD and 34.29 for A-CUD. The most prescribed drugs for AUD were diazepam (54.1 %), thiamine (37 %), complex B vitamins (29.5 %), and disulfiram (2.7 %); for CUD, diazepam (26.9 %) and haloperidol (23.1 %). It should be noticed that although contraindicated by the guidelines, chlorpromazine (42.3 %, 25.3 %, 20.3 %) was prescribed for CUD, AUD, and A-CUD respectively. Knowing the pharmacoepidemiological profile of CAPS-AD patients is extremely important for making decisions regarding which medicines to make available to the population.

Genetic Determinants for Prediction of Outcome of Patients with Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) usually presents an excellent prognosis, but some patients present with aggressive metastatic disease. BRAF, RAS, and TERT promoter (TERTp) genes are altered in PTC, and their impact on patient outcomes remains controversial. We aimed to determine the role of genetic alterations in PTC patient outcomes (recurrent/persistent disease, structural disease, and disease-specific mortality (DSM)). The series included 241 PTC patients submitted to surgery, between 2002-2015, in a single hospital. DNA was extracted from tissue samples of 287 lesions (primary tumors and metastases). Molecular alterations were detected by Sanger sequencing. Primary tumors presented 143 BRAF, 16 TERTp, and 13 RAS mutations. Isolated TERTpmut showed increased risk of structural disease (HR = 7.0, p < 0.001) and DSM (HR = 10.1, p = 0.001). Combined genotypes, BRAFwt/TERTpmut (HR = 6.8, p = 0.003), BRAFmut/TERTpmut (HR = 3.2, p = 0.056) and BRAFmut/TERTpwt (HR = 2.2, p = 0.023) showed increased risk of recurrent/persistent disease. Patients with tumors BRAFwt/TERTpmut (HR = 24.2, p < 0.001) and BRAFmut/TERTpmut (HR = 11.5, p = 0.002) showed increased risk of structural disease. DSM was significantly increased in patients with TERTpmut regardless of BRAF status (BRAFmut/TERTpmut, log-rank p < 0.001; BRAFwt/TERTpmut, log-rank p < 0.001). Our results indicate that molecular markers may have a role in predicting PTC patients' outcome. BRAFmut/TERTpwt tumors were prone to associate with local aggressiveness (recurrent/persistent disease), whereas TERTpmut tumors were predisposed to recurrent structural disease and DSM.

Potentially inappropriate medication use in older adults: prevalence and physician knowledge / Medicamentos potencialmente inapropriados para idosos: prevalência de uso e conhecimento médico

OBJECTIVE: To analyze the frequency, profile, and additional variables associated with the prescription of potentially inappropriate medications (PIM) to older adults in primary care, and evaluate physicians' knowledge about these medications. METHODS: A cross-sectional study was conducted based on data from patient records for the period of January 2014 to December 2017 in a city located in the state of Minas Gerais. The frequency of PIM use was evaluated based on the 2019 Beers-Fick criteria. Physician knowledge was evaluated using a validated questionnaire as a primary data source. RESULTS: In a sample of 423 older adults, 75.89% (n = 321) used at least one PIM, the most common of which were medications used to treat central nervous system disorders (48.00%; n = 203). Most participants were female (62.41%; n = 264) and 70 years or older (69.50%; n = 294). When presented with clinical cases illustrating common situations in the management of older patients, 53.33% of physicians (n = 8) answered four or five questions correctly out of a possible seven; 13.33% (n = 2) answered six questions correctly; and 33.33% (n = 5) obtained three correct answers or fewer. CONCLUSIONS: These findings showed a high frequency of PIM use among older adults treated in Primary Health Care settings, with medications used in the treatment of central nervous system disorders. Our results highlight the importance of continuing education for health professionals and improved assessments of the medication available in the Unified Health System (Sistema Único de Saúde; SUS) for use in older adults, especially those taking multiple medications.

OBJETIVOS: Analisar a frequência, o perfil e os fatores associados à prescrição de medicamentos potencialmente inapropriados (MPI) aos idosos na Atenção Primária à Saúde, além de avaliar o conhecimento dos médicos sobre esses medicamentos. METODOLOGIA: Trata-se de um estudo transversal, cujas informações foram coletadas de prontuários dos pacientes, referentes ao período de janeiro de 2014 a dezembro de 2017, em um município localizado no estado de Minas Gerais. Para análise da frequência de uso de MPI, utilizou-se o critério Beers-Fick de 2019. Para avaliação do conhecimento médico, utilizou-se uma fonte primária de informação, um questionário validado. RESULTADOS: Dos 423 idosos, 75,89% (n=321) utilizavam pelo menos um MPI e a classe mais utilizada foi do sistema nervoso central (48,00%; n=203). Houve maior prevalência no sexo feminino (62,41%; n=264) e em idosos com idade ≥70 anos (69,50%; n=294). Em relação aos casos clínicos apresentados para avaliar o conhecimento dos médicos referentes às situações comuns no cuidado à saúde do idoso, 53,33% (n=8) acertaram quatro ou cinco questões das sete existentes, enquanto 13,33% (n=2) responderam corretamente a seis perguntas e 33,33% (n=5) obtiveram três ou menos acertos. CONCLUSÕES: Os resultados evidenciaram alta taxa de prescrição de MPI entre os idosos da Atenção Primária à Saúde, destacando-se os medicamentos que são destinados ao tratamento de doenças do sistema nervoso central. Ressalta-se a importância da educação continuada desses profissionais de saúde e a melhor avaliação de medicamentos incorporados ao Sistema Único de Saúde (SUS) e destinados à população geriátrica, sobretudo o idoso polimedicado.

Bulas de medicamentos cardiovasculares: elas têm informações sobre precauções necessárias aos idosos? / Cardiovascular drug labeling: Do they have information on necessary precautions for older people?

OBJETIVO: Analisar a existência de informações, em bulas destinadas aos profissionais de saúde, sobre precaução de uso de medicamentos cardiovasculares em idosos. METODOLOGIA: Trata-se de estudo documental realizado por meio de análise de dados contidos em bulas de medicamentos. Analisou-se a existência, na bula, de informações sobre precauções no uso de medicamentos para idosos, conforme descrito no Consenso Brasileiro de Medicamentos Potencialmente Inapropriados para Idosos de 2016. RESULTADOS: Dos 29 medicamentos pertencentes ao grupo do sistema cardiovascular que devem ser evitados por idosos e estão disponíveis no Brasil, 15 independem da condição clínica prévia do paciente para que sejam vetados para os idosos. Desses 15, apenas 3 medicamentos (20%) têm informações explícitas concordantes com o Consenso (metildopa, digoxina e espironolactona); 2 (13,33%) têm informações explícitas ausentes; 4 (26,66%) têm informações explícitas discordantes; e 6 bulas (40%) foram categorizadas como informações não explícitas. Quanto às precauções dos medicamentos de acordo com a condição clínica do paciente, incluíram-se 14 medicamentos que devem ser evitados por idosos e estão disponíveis no Brasil. Destes, 12 (85,71%) têm em suas bulas recomendações concordantes com o Consenso, porém não explícitas, e 2 (14,28%) não têm as contraindicações. CONCLUSÕES: A maioria das bulas carece de informações sobre precauções de uso de medicamentos para a população idosa.

OBJECTIVE: To analyze the existence of information on drug labeling intended for health professionals on the precaution of cardiovascular drugs use in older people. METHODS: This is a documentary study, carried out with the analysis of data contained in drug labelings. The existence of information on precautions in the use of drugs for older people as described in the 2016 Brazilian Consensus on Potentially Inappropriate Drugs for Older People (Consenso Brasileiro de Medicamentos Potencialmente Inapropriados para Idosos de 2016). RESULTS: Of the 29 drugs belonging to the cardiovascular system group that should be avoided by older people and are available in Brazil, 15 are independent of the clinical condition. Of these fifteen, only three drugs (20%) have explicit information in accordance with the Consensus (methyldopa, digoxin, and spironolactone); two (13.33%) have missing explicit information; four (26.66%) have explicitly discordant information; and six drug labels (40%) were categorized as non-explicit information. Regarding drug precautions according to clinical condition, 14 drugs were included. Of these, 12 (85.71%) have equal contraindications of that of Consensus on their drug labels, however, non-explicit; and two (14.28%) contraindications are missing. CONCLUSION: Most drug labels lack information on the precautions for the use of drugs in older people.

Comprehensive Assessment of TERT mRNA Expression across a Large Cohort of Benign and Malignant Thyroid Tumours.

The presence of TERT promoter (TERTp) mutations in thyroid cancer have been associated with worse prognosis features, whereas the extent and meaning of the expression and activation of TERT in thyroid tumours is still largely unknown. We analysed frozen samples from a series of benign and malignant thyroid tumours, displaying non-aggressive features and low mutational burden in order to evaluate the presence of TERTp mutations and TERT mRNA expression in these settings. In this series, TERTp mutations were found in 2%, only in malignant cases, in larger cancers, and from older patients. TERT mRNA expression was detected in both benign and malignant tumours, with increased frequencies in the malignant tumours with aggressive histotypes, larger tumours, and from older patients. In benign tumours, TERT mRNA expression was found in 17% of the follicular thyroid adenoma (FTA) with increased levels of expression in smaller tumours and associated with the presence of thyroiditis. TERTp mutations and TERT mRNA expression are correlated with worse prognosis features in malignant thyroid tumours, whereas TERT mRNA expression in the benign tumours is associated with the presence of thyroiditis.

Prognostic Significance of RAS Mutations and P53 Expression in Cutaneous Squamous Cell Carcinomas.

TP53 is considered the most commonly-altered gene in cutaneous squamous cell carcinoma (cSCC). Conversely, RAS mutations have been reported in a low percentage of cSCC. The objective of our study was to evaluate the frequency of p53 expression and RAS mutations in cSCC and correlate them with clinicopathological features and patient outcome. We performed immunohistochemistry for p53 and genetic profiling for RAS mutations in a retrospective series of cSCC. The predictive value of p53 expression, RAS mutations, and clinicopathological parameters was assessed using logistic regression models. The overall frequency of RAS mutations was 9.3% (15/162), and 82.1% of the cases (133/162) had p53 overexpression. RAS mutations rate was 3.2% (1/31) of in situ cSCCs and 10.7% (14/131) of invasive cSCCs. RAS mutations were more frequently associated with an infiltrative than an expansive pattern of invasion (p = 0.046). p53 overexpression was a predictor of recurrence in the univariate analysis. Our results indicate that RAS mutations associate with features of local aggressiveness. Larger studies with more recurrent and metastatic cSCCs are necessary to further address the prognostic significance of p53 overexpression in patients' risk stratification.

Evaluation of the role of mitochondria in the non-targeted effects of ionizing radiation using cybrid cellular models.

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. The mitochondrial role in orchestrating this biological response needs to be further explored. Cybrids (cytoplasmic hybrids) are useful cell models for studying the involvement of mitochondria in cellular processes. In the present study we used cybrid cell lines to investigate the role of mitochondria in the response to radiation exposure. Cybrid cell lines, derived from the osteosarcoma human cell line 143B, harboring, either wild-type mitochondrial DNA (Cy143Bwt), cells with mitochondria with mutated DNA that causes mitochondrial dysfunction (Cy143Bmut), as well as cells without mitochondrial DNA (mtDNA) (143B-Rho0), were irradiated with 0.2 Gy and 2.0 Gy. Evaluation of the non-targeted (or bystander) effects in non-irradiated cells were assessed by using conditioned media from the irradiated cells. DNA double stranded breaks were assessed with the γH2AX assay. Both directly irradiated cells and cells treated with the conditioned media, showed increased DNA damage. The effect of the irradiated cells media was different according to the cell line it derived from: from Cy143Bwt cells irradiated with 0.2 Gy (low dose) and from Cy143Bmut irradiated with 2.0 Gy (high dose) induced highest DNA damage. Notably, media obtained from cells without mtDNA, the143B-Rho0 cell line, produced no effect in DNA damage. These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. Furthermore, it indicates that cybrid models are valuable tools for radiobiological studies.

Interaction of Genetic Variations in NFE2L2 and SELENOS Modulates the Risk of Hashimoto's Thyroiditis.

Background: Several single-nucleotide polymorphisms (SNPs) are known to increase the risk of Hashimoto's thyroiditis (HT); such SNPs reside in thyroid-specific genes or in genes related to autoimmunity, inflammation, and/or cellular defense to stress. The transcription factor Nrf2, encoded by NFE2L2, is a master regulator of the cellular antioxidant response. This study aimed to evaluate the impact of genetic variation in NFE2L2 on the risk of developing HT. Methods: In a case-control candidate gene association study, functional SNPs in the NFE2L2 promoter (rs35652124, rs6706649, and rs6721961) were examined either as independent risk factors or in combination with a previously characterized HT risk allele (rs28665122) in the gene SELENOS, encoding selenoprotein S (SelS). A total of 997 individuals from the north of Portugal (Porto) were enrolled, comprising 481 HT patients and 516 unrelated healthy controls. SELENOS and NFE2L2 SNPs were genotyped using TaqMan® assays and Sanger sequencing, respectively. Odds ratios (ORs) were calculated using logistic regression, with adjustment for sex and age. Expression of SelS was analyzed by immunohistochemistry in thyroid tissue from HT patients and control subjects. Molecular interactions between the Nrf2 and SelS pathways were investigated in thyroid tissues from mice and in rat PCCL3 thyroid follicular cells. Results: When all three NFE2L2 SNPs were considered together, the presence of one or more minor alleles was associated with a near-significant increased risk (OR = 1.43, p = 0.072). Among subjects harboring only major NFE2L2 alleles, there was no increased HT risk associated with heterozygosity or homozygosity for the SELENOS minor allele. Conversely, in subjects heterozygous or homozygous for the SELENOS risk allele, the presence of an NFE2L2 minor allele significantly increased HT risk by 2.8-fold (p = 0.003). Immunohistochemistry showed reduced expression of SelS in thyroid follicular cells of HT patients. In Nrf2 knockout mice, there was reduced expression of SelS in thyroid follicular cells; conversely, in PCCL3 cells, reducing SelS expression caused reduced activity of Nrf2 signaling. Conclusions: The NFE2L2 promoter genotype interacts with the SELENOS promoter genotype to modulate the risk of HT in a Portuguese population. This interaction may be due to a bidirectional positive feedback between the Nrf2 and SelS pathways.

TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma.

BACKGROUND: Telomerase reverse transcriptase gene (TERT) promoter (TERTp) mutations have been reported as potential predictors of poor prognosis in several cancers, but the prognostic value of TERTp mutations for cutaneous squamous cell carcinoma (cSCC) has not been determined. OBJECTIVE: To evaluate the frequency of TERTp mutations and correlate it with clinicopathologic features and patient outcome. METHODS: We performed genetic profiling of TERTp mutations in a retrospective series of cSCCs. The predictive value of TERTp mutations and clinicopathologic parameters were assessed by using logistic regression models. RESULTS: A total of 152 cSCCs from 122 patients were analyzed for TERTp mutations; the mutation rate was 31.6% (48 of 152), and it was higher in invasive cSCC (42 of 121 [34.7%]) than in in situ cSCC (6 of 31 [19.4%]). Age older than 75 years (odds ratio [OR], 14.84; P = .013] and TERTp mutation (OR, 8.11; P = .002) were independent predictors of local recurrence. TERTp mutation (OR, 15.89; P = .022) was independently associated with higher risk of lymph node metastasis. LIMITATIONS: The restricted number of metastatic cases. CONCLUSION: TERTp mutations may prove to be a molecular biomarker with prognostic significance in invasive cSCC, but larger studies are needed.

OPNa Overexpression Is Associated with Matrix Calcification in Thyroid Cancer Cell Lines.

Osteopontin (OPN) spliced variants (OPN-SV: OPNa, OPNb, and OPNc) are aberrantly expressed in tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer (TC). PTC often presents with desmoplasia and dystrophic calcification, including psammoma bodies (PB). This work aimed to investigate total OPN (tOPN) and OPN-SV expression and their association with the presence of PB in the PTC classical variants (cPTC), as well as the involvement of OPN-SV in matrix calcification of TC cell lines. We found that cPTC samples presenting PB showed higher OPN expression levels. In TC cell lines, OPNa overexpression promotes higher matrix calcification and collagen synthesis when compared to that of clones overexpressing OPNb or OPNc. In response to OPN knockdown, calcification was inhibited, paralleled with the downregulation of calcification markers. In conclusion, our data evidenced that OPN expression is associated with the presence of PB in cPTC samples. Among the OPN-SV, OPNa is the main contributor to matrix calcification in tested TC cells, providing clues to a better understanding on the biology and ethiopathogenesis of the calcification process in TC cells.

TERTp mutation is associated with a shorter progression free survival in patients with aggressive histology subtypes of follicular-cell derived thyroid carcinoma.

PURPOSE: Evaluate the impact of TERTp mutation on the outcomes after initial treatment of 45 patients with thyroid carcinomas derived from follicular cells (TCDFC) with aggressive histology, in which the role of this mutation is not yet well defined. METHODS: Analysis of the presence of TERTp (-124C > T and -146C > T), BRAF (V600E), and NRAS (Q 61R) mutations by Sanger sequencing and analysis of their correlation with the patient's outcomes. RESULTS: Forty-five patients with aggressive histopathologic variants were included in the study. Of these, 68.9% had aggressive variants of papillary thyroid cancer (PTC), 22.2% had poorly differentiated thyroid carcinoma (PDTC)/insular carcinoma, and 8.9% had invasive follicular thyroid cancer (FTC) with Hurthle cell features (Hurthle cell carcinoma). Lymph node metastases were present in 46.7% and distant metastases in 54.6%. The response to the initial therapy was excellent in 45.5% and structurally incomplete in 50%. During the follow-up period (median of 56 months; 5-360 months), 47.7% presented with disease progression and 17.8% experienced disease-related death. In 53.3% of the cases at least one molecular alteration (TERTp in 33.4%, BRAF in 24.5%, RAS in 8.9%) was detected. In the multivariate analysis, TERTp mutation was the factor associated with the highest risk (6 times) of having structural disease after initial therapy (p = 0.01), followed by vascular invasion (p = 0.02), gross extrathyroidal extension (ETE) (p = 0.02) and distant metastasis (p = 0.04). Regarding mutational status, only TERTp mutation was associated with disease progression, and diminished disease progression-free survival (PFS). The presence of distant metastasis, vascular invasion and gross ETE were significantly associated with the risk of disease progression. CONCLUSIONS: TERTp mutation appears be an indicator of both persistence and progression of structural disease after initial therapy in aggressive variants of TCDFC, and associates with a shorter progression free survival regardless of the therapy employed.

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression.

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

CRABP1, C1QL1 and LCN2 are biomarkers of differentiated thyroid carcinoma, and predict extrathyroidal extension.

BACKGROUND: The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis. METHODS: We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers. RESULTS: We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC. CONCLUSIONS: We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.

NIS expression in thyroid tumors, relation with prognosis clinicopathological and molecular features.

Thyroid cancer therapy is based on surgery followed by radioiodine treatment. The incorporation of radioiodine by cancer cells is mediated by sodium iodide symporter (NIS) (codified by the SLC5A5 gene), that is functional only when targeted to the cell membrane. We aimed to evaluate if NIS expression in thyroid primary tumors would be helpful in predicting tumor behavior, response to therapy and prognosis. NIS expression was addressed by qPCR and immunohistochemistry. In order to validate our data, we also studied SLC5A5 expression on 378 primary papillary thyroid carcinomas from The Cancer Genome Atlas (TCGA) database. In our series, SLC5A5 expression was lower in carcinomas with vascular invasion and with extrathyroidal extension and in those harboring BRAFV600E mutation. Analysis of SLC5A5 expression from TCGA database confirmed our results. Furthermore, it showed that larger tumors, with locoregional recurrences and/or distant metastases or harboring RAS, BRAF and/or TERT promoter (TERTp) mutations presented significantly less SLC5A5 expression. Regarding immunohistochemistry, 12/211 of the cases demonstrated NIS in the membrane of tumor cells, those cases showed variable outcomes concerning therapy success, prognosis and all but one were wild type for BRAF, NRAS and TERTp mutations. SLC5A5 mRNA lower expression is associated with features of aggressiveness and with key genetic alterations involving BRAF, RAS and TERTp. Mutations in these genes seem to decrease protein expression and its targeting to the cell membrane. SLC5A5 mRNA expression is more informative than NIS immunohistochemical expression regarding tumor aggressiveness and prognostic features.

TERT biology and function in cancer: beyond immortalisation.

Evasion of replicative senescence and proliferation without restriction, sometimes designated as immortalisation, is one of the hallmarks of cancer that may be attained through reactivation of telomerase in somatic cells. In contrast to most normal cells in which there is lack of telomerase activity, upregulation of TERT transcription/activity is detected in 80-90% of malignant tumours. In several types of cancer, there is a relationship between the presence of TERT promoter mutations, TERT mRNA expression and clinicopathological features, but the biological bridge between the occurrence of TERT promoter mutations and the aggressive/invasive features displayed by the tumours remains unidentified. We and others have associated the presence of TERT promoter mutations with metastisation/survival in several types of cancer. In follicular cell-derived thyroid cancer, such mutations are associated with worse prognostic features (age of patients, tumour size and tumour stage) as well as with distant metastases, worse response to treatment and poorer survival. In this review, we analyse the data reported in several studies that imply TERT transcription reactivation/activity with cell proliferation, tumour invasion and metastisation. A particular attention is given to the putative connections between TERT transcriptional reactivation and signalling pathways frequently altered in cancer, such as c-MYC, NF-κB and B-Catenin.