Método e kit para predição prognóstico de câncer e uso do método em oncologia clínica

Universidade Federal do Rio Grande do SulMedicinaDepositad

Intraoperative frozen section performance for thyroid cancer diagnosis

Objective: A primary medical relevance of thyroid nodules consists of excluding thyroid cancer, present in approximately 5% of all thyroid nodules. Fine-needle aspiration biopsy (FNAB) has a paramount role in distinguishing benign from malignant thyroid nodules due to its availability and diagnostic performance. Nevertheless, intraoperative frozen section (iFS) is still advocated as a valuable tool for surgery planning, especially for indeterminate nodules. Subjects and methods: To compare the FNAB and iFS performances in thyroid cancer diagnosis among nodules in Bethesda Categories (BC) I to VI. The performance of FNAB and iFS tests were calculated using final histopathology results as the gold standard. Results: In total, 316 patients were included in the analysis. Both FNAB and iFS data were available for 272 patients (86.1%). The overall malignancy rate was 30.4%% (n = 96). The FNAB sensitivity, specificity, and accuracy for benign (BC II) and malignant (BC V and VI) were 89.5%, 97.1%, and 94.1%, respectively. For all nodules evaluated, the iFS sensitivity, specificity, and accuracy were 80.9%, 100%, and 94.9%, respectively. For indeterminate nodules and follicular lesions (BC III and IV), the iFS sensitivity, specificity, and accuracy were 25%, 100%, and 88.7%, respectively. For BC I nodules, iFS had 95.2% of accuracy. Conclusion: Our results do not support routine iFS for indeterminate nodules or follicular neoplasms (BC III and IV) due to its low sensitivity. In these categories, iFS is not sufficiently accurate to guide the intraoperative management of thyroidectomies. iFS for BC I nodules could be an option and should be specifically investigated

Type 2 deiodinase Thr92Ala polymorphism is not associated with cognitive impairment in older adults : a cross-sectional study

Background: Type 2 Deiodinase (DIO2) converts thyroxine (T4) into the active hormone triiodothyronine (T3). Thr92Ala DIO2 polymorphism has been associated with reduced conversion of T4 into T3 and central nervous system hypothyroidism. However, how Thr92Ala DIO2 polymorphism affects cognitive function is still unclear. Objective: To assess the association between Thr92Ala DIO2 polymorphism and cognitive performance in older adults. Design: Cross-sectional study. Setting: University-based tertiary hospital in Brazil. Patients: > 65-year-old with no limiting clinical disease. Interventions: All participants answered a standard questionnaire before undergoing thyroid function laboratory evaluation and genotyping of the Thr92Ala DIO2 polymorphism. Main Outcomes: Cognitive impairment measured by the Word List Memory task from the Consortium to Establish a Registry for Alzheimer’s Disease Neuropsychological Battery (CERAD-NB) and the Brief Cognitive Screening Battery (BCSB). Results: A hundred individuals were included. Clinical and laboratory characteristics were similar among DIO2 genotypes (all p > 0.05). No differences were found in the Word List Memory, recall, or recognition tests of the CERAD-NB assuming a recessive model for the Ala/Ala vs. Thr/Ala-Thr/Thr genotypes. Results of Clock Drawing Test, Animal Fluency Test, Mini-Mental State Exam, and Figure Memory Test of the BCSB were similar between groups. Conclusions: These findings suggest that Thr92Ala DIO2 polymorphism is not associated with relevant cognitive impairment in older adults

Expressão e regulação da enzima desiodase tipo 3 em neoplasias mamárias

O câncer de mama é uma doença altamente heterogênea, sendo que a identificação de biomarcadores que predigam o comportamento biológico do tumor contribuem para definição do prognóstico e estratégica terapêutica. Os hormônios tireoidianos (HT) são reguladores essenciais de diversos processos celulares, e alterações no status do HTs interferem na progressão tumoral através de virtualmente todos os marcos do câncer (“hallmarks of cancer”). Estudos clínicos têm associado os níveis de HTs a risco de desenvolvimento de câncer de mama, enquanto estudos in vitro têm demonstrado que os HTs influenciam a proliferação, apoptose e migração de células tumorais mamárias. A enzima desiodase tipo 3 (DIO3) é a principal enzima na inativação dos hormônios tireoidianos, e alterações na expressão dessa enzima tem sido descritas em diversas neoplasias humanas. Na primeira parte desta tese, o leitor encontrará um artigo de revisão sobre o papel dos hormônios tireoidianos no processo neoplásico e seus efeitos sobre cada hallmark do câncer. Na segunda parte, é apresentado um levantamento de dados originais e revisão sobre a expressão das desiodases - enzimas que ativam e inativam os hormônios tireoidianos – em diferentes neoplasias humanas, e seu potencial efeito sobre o processo tumoral. Na terceira parte, é apresentado o artigo original desta tese, com objetivos, metodologia, resultados e discussão dos mesmos. O objetivo deste trabalho foi avaliar a expressão e valor prognóstico da DIO3 em câncer de mama em humanos. Para isso foram utilizadas duas coortes retrospectivas de pacientes com câncer de mama. A expressão da enzima DIO3 foi avaliada através de técnica de imunohistoquímica em tecido de mama de 53 pacientes e quantificada através de H-Score em uma coorte primária. Subsequentemente, os resultados foram validados em uma segunda coorte de 1094 pacientes com câncer de mama utilizando-se dados de RNA sequencing (RNA-Seq) da base de dados The Cancer Genome Atlas (TCGA). Em ambas as populações, os dados de expressão foram correlacionados com dados clínico-patológicos dos pacientes, a significância prognóstica da expressão da enzima foi avaliada através de regressão de Cox e a avaliação de sobrevida foi realizada por método de Kaplan-Meier. O padrão de metilação de DNA da região genômica do gene DIO3 em mama foi analisado utilizando-se dados clínicos e de metilação de DNA de 890 pacientes provenientes da base de dados do TCGA. Adicionalmente, a regulação da enzima foi avaliada em linhagens celulares derivadas de câncer de mama (células MCF-7 e MDA-MB-231). 13 A expressão proteica de DIO3 foi encontrada em 35/39 (89.7%) das amostras de carcinoma ductal invasor, com H-Score médio de 104.9 ± 55, e em apenas uma amostra de três analisadas de carcinoma lobular invasor (H-Score=86). O mRNA do gene DIO3 está expresso em tecido mamário normal e tumoral, com expressão de mRNA reduzida em tumores em relação a tecido normal (logFC =-1.54, P ajustado <0.00001). A intensidade de expressão de DIO3 não se correlacionou com características clínico-patológicas dos pacientes na coorte primária, como tamanho tumoral, presença de metástase linfonodal ou à distância, positividade para receptor de estrógeno (RE), receptor de progesterona (RP) ou receptor epidérmico humano 2 do fator de crescimento (HER2). Entretanto, na mesma coorte, em análise univariada utilizando-se mortalidade como desfecho primário, a negatividade para expressão da DIO3 se associou a maior risco de morte (HR 4.29 [IC 95%, 1.24-14.7] P=0.021), sendo que pacientes com ausência de expressão de DIO3 tiveram menor sobrevida em relação à pacientes que expressavam DIO3 (73.3 meses [IC 95%, 41 a 105] vs. 122 meses [IC 95%, 109 a 135]; log-rank P=0.012). Validamos estes achados na segunda coorte (N=1094), onde a baixa expressão do gene DIO3 se correlacionou com maior tamanho tumoral (P=0.019) e negatividade para RE (P=0.022). Confirmando os achados da coorte primária, baixa expressão de DIO3 se associou a menor sobrevida global (HR 1.6 [IC 95% 1.18-2.26]; P=0.003) em modelo univariável e se manteve como preditor independente de prognóstico em modelo multiváriavel ajustado para idade, tamanho tumoral, presença de metástase linfonodal e à distância, status de RE e RP (HR 1.55 [IC 95% 1.07-2.24]; P=0.02). A sobrevida global em 5 anos foi de 90.4% (IC 95%, 86.4%-94.5%) no grupo com alta expressão de DIO3 e 77.4% (IC 95%, 71.3%-84.1%) no grupo com baixa expressão. A análise de metilação de DNA revelou que a região do gene DIO3 encontra-se hipermetilada em tecido tumoral relação ao tecido normal (p<0.0001), em especial os sítios CpGs localizados na região promotora do gene. A análise da regulação de DIO3 em linhagem celulares MCF-7 e MDA-MB-231 demonstrou indução do mRNA de DIO3 quando ambas as linhagens celulares foram submetidas a tratamento com 10 nM de triiodotironina (T3) por 24h. Além disso, ocorreu inibição dose-dependente do mRNA quando as células MCF-7 foram tratadas com dexametasona em doses de 10 e 100 nM, efeito que não se observou em células MDA-MB-231. A inibição da via mitogen-activated protein kinase (MAPK) com utilização do inibidor MEK-específico U0126 (10 uM) levou à redução de 50% na expressão de mRNA de DIO3 (P=0.004) em células MCF-7. 14 Em conclusão, nossos resultados indicam que a enzima DIO3 encontra-se expressa em tecido mamário normal e em câncer de mama. De modo interessante, a diminuição ou perda expressão de DIO3/DIO3 foi fator independente para menor sobrevida em duas populações distintas. A redução da expressão da DIO3 em câncer de mama pode ser explicada ao menos em parte por hipermetilação da região promotora do gene neste tipo tumoral. Em linhagem celular MCF-7, a enzima mantém suas características de regulação pré-transcricional por T3, dexametasona e modulação pela via da MAPK. Esses resultados apontam para a DIO3 como marcador prognóstico em câncer de mama, sendo a redução de sua expressão associada a pior sobrevida.Breast cancer is a highly heterogeneous disease and the identification of biomarkers that predict tumor biological behavior is warranted in improving patient survival. Thyroid hormones (THs) are critical regulators of cellular processes, and TH status alterations are known to contribute to cancer progression through all the hallmarks of cancer. Clinical studies associate THs levels with breast cancer mortality, and THs have been shown to influence breast cancer proliferation, apoptosis, and migration in in vitro models. Type 3 deiodinase (DIO3) is the main enzyme responsible for TH inactivation and disturbed DIO3 expression has been demonstrated in several human neoplasias. In the first part of this thesis, the reader will find a review article concerning the role of the thyroid hormones in the neoplastic process and their effect on each hallmark of cancer. In the second part, we present original data and a review on current evidence of deiodinases – enzymes that activate and inactivate thyroid hormones - expression in human neoplasias, as well as their potential role in the neoplastic process. In the third part, we present the main aim of this thesis, our methods, results, and their discussion. We aimed to evaluate expression patterns and the prognostic significance of DIO3 in breast cancer in humans. The expression of DIO3 was evaluated through immunohistochemistry in a primary cohort of 53 samples of breast tissue and quantified by the H-Score method. Subsequently, these results were validated in a second cohort of 1094 patients using the RNA sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) database. We assessed DIO3 expression in both populations according to retrieved clinicopathological information. The prognostic value of DIO3 expression was evaluated through Cox regression analysis, and survival analysis was assessed by the Kaplan-Meier method. DNA methylation and clinical data for 890 samples from the TCGA study were obtained to evaluate levels of methylation of the DIO3 gene region in breast cancer. We also evaluated DIO3 regulation in breast cancer cell lines MCF-7 and MDA-MB-231. DIO3 protein expression was present in both normal and tumoral breast glandular tissue. DIO3 expression in FFPE tissues of breast cancer was positive in 35/39 (89.7%) of Invasive Ductal Carcinoma (IDC), with a mean H-Score of 104.9 ± 55, and only in 1 of 3 samples of invasive lobular carcinoma (ILC) (H-Score=86). DIO3 mRNA expression was found to be reduced in tumor samples when compared to healthy tissue, (logFC =-1.54, adjusted P<0.0001). DIO3 staining intensity did not correlate with clinicopathologic 16 characteristics in the primary cohort such as tumor size, the presence of lymph node or distant metastasis, estrogen or progesterone receptor positivity or HER2 positivity. However, the univariate analysis with overall survival (OS) as the primary outcome, loss of DIO3 expression was associated with increased mortality (HR 4.29 [95% CI, 1.24-14.7] P=0.021). Patients with negative DIO3 expression had worse OS than those positive DIO3 expression (73.3 months [95% CI, 41 to 105)] vs. 122 months [95% CI, 109 to 135]; log-rank P=0.012). We then validated this finding in the second cohort (N=1094). Interestingly, low DIO3 expression was associated with greater tumor size (P=0.019) and estrogen receptor negativity (P=0.022), Confirming our results in the primary cohort, low DIO3 expression was associated with worse overall survival in a univariate model (HR 1.6 [95% CI, 1.18-2.26]; P=0.003) and remained as an independent prognostic factor in a multivariate model adjusted for age, tumor size, lymph node and distant metastasis, ER and PR status (HR 1.55 [95% CI, 1.07-2.24]; P=0.02). The estimated rate of overall survival at five years in the Kaplan–Meier analysis was 90.4% (95% CI, 86.4% - 94.5%) in the high DIO3 group and 77.4% (95% CI, 71.3% - 84.1%) in the low DIO3 group. DNA methylation analysis revealed that DIO3 gene promoter is hypermethylated in tumoral samples when compared to normal tissue (p <0.0001). Additional experiments were performed to determine DIO3 regulation in breast cancer cells. In MCF-7 and MDA-MB-231 cells, DIO3 was subject to T3 stimulation (10 nM). We observed a dose-dependent inhibition of DIO3 when MCF-7 cells were treated with dexamethasone 10 and 100 nM, an effect that was not observed in MDA-MB-231 cells. Also in MCF-7 cells, mitogen-activated protein kinase (MAPK) pathway inhibition using specific MEK inhibitor U0126 (10 uM) resulted in 50% reduction of DIO3 expression (P=0.004). In conclusion, our results demonstrate that DIO3 is expressed in normal and tumoral breast tissue. We showed that low DIO3 expression was an independent factor associated with reduced overall survival in two different populations of breast cancer. Loss of DIO3 expression in breast cancer can be explained at least in part by hypermethylation of the promoter region of the gene. The enzyme maintains its regulation by T3, dexamethasone and it is subject to MAPK modulation in MCF-7 cells. In summary, our results point to DIO3 as a new prognostic marker in breast cancer, as loss of its expression is associated with reduced overall survival

Rectal prolapse repair via vaginal route

Many elderly patients have concomitant anorectal, urological and gynecological symptoms involving multiple perineal structures, which require the surgeon to operate in different areas of his specialty in order to correct different dystopias in a single procedure. We describe a technique for total rectal prolapse correction offering low surgical risk for patients presenting with this pathology along with pelvic floor defects. We used a simple, minimally invasive technique, which showed complete success in the postoperative follow-up. The patients showed no peri- and post-operative complications and were discharged briefly after the procedure. Although it was a small sample, there was no case of rectal prolapse recurrence with the technique described. With the procedure described here, the treatment of rectal prolapse is possible with concomitant pelvic reconstruction in a single procedure, which is an interesting approach for elderly patients commonly affected by both diseases, as these patients often have other comorbidities that increase the perioperative ris