Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma

© The Author(s). 2021 Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Background: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.info:eu-repo/semantics/publishedVersio

Composição e abundância de macrófitas num troço do rio Ovelha

As macrófitas fluviais são um grupo relevante para a avaliação ecológica dos rios. Numa amostragem realizada num troço de 100 m do rio Ovelha, localizado a 217 m de altitude, na freguesia de Fornos, Marco de Canaveses, estudou-se a abundância, composição e distribuição das macrófitas. Verificou-se que o troço estudado é pobre em macrófitas, apresentando uma riqueza específica baixa, o que poderá estar relacionado, sobretudo, com o substrato rochoso. Considerando os resultados obtidos é fundamental que, futuramente, sejam estudadas as macrófitas conjuntamente com outros elementos biológicos, no sentido de se proceder a uma correta monitorização do estado ecológico do rio Ovelha.info:eu-repo/semantics/publishedVersio

Phenotypical and molecular characterization of portuguese leber congenital amaurosis patients

Trabalho final de mestrado integrado em Medicina àrea científica de Oftalmologia, apresentado à Faculdade de Medicina da Universidade de CoimbraIntroduction: Leber Congenital Amaurosis encompasses a group of early onset retinal dystrophies causing severe visual impairment, nystagmus and retinal dysfunction. It is mostly an autosomal recessive condition and to date 19 genes have been identified as potential culprits. Our aim is to characterize in a molecular and phenotypical standpoint, 28 affected Portuguese patients, determine if they carry mutations in the known genes and establish potential genotype-phenotype correlations both with respect to retinal structural and functional changes. Methods: Twenty eight individuals from 26 unrelated families (twelve males, sixteen females) were characterized by clinical examination, electrophysiology (ERG), mutation analysis, optical coherence tomography (OCT), autofluorescence, head MRI and renal function testing. Results: LCA was demonstrated in all patients. Consanguinity could be documented in 25% of families. Clinically, patients complained of nyctalopia in 21% of cases and the typical oculo-digital sign of Franceschetti was observed in only 18% of cases. High hyperopia was the most prevalent refractive error. In our cohort the fundus appearance varied from anatomically normal (4%), non-specific changes/atrophy of the retinal pigment epithelium (RPE) (25%), peripheral pigmented changes (64%) and macular coloboma-like defects (25%). We observed 29% of cases with some degree of developmental delay and 21% with clear signs that fit criteria of the autism/autistic behaviour spectrum. Molecular testing is still an ongoing process; thus far, causative mutations in the known LCA genes have been identified in 5 independent cases with the NPHP6 gene being mutated in 3 patients, the RPGRIP1 gene in one patient and the NPHP5 in another patient. The NPHP5 patient was later reclassified as Senior-Loken syndrome. Our results fit those found in international literature. Conclusion: We characterize from a clinical and genetic standpoint, the largest series of Portuguese patients with LCA. In-depth knowledge of this group of conditions is invaluable for appropriate counselling and possibly treatment, in the near futureIntrodução: A Amaurose Congénita de Leber abrange um grupo de distrofias retinianas de aparecimento precoce que causam baixa de visão grave e disfunção retiniana. É uma condição maioritariamente autossómica recessiva e, até à data, 19 genes foram identificados como possíveis causadores desta doença. O nosso objectivo neste trabalho é caracterizar molecular e fenotipicamente 28 doentes Portugueses, determinar se são portadores de mutações nos genes conhecidos e estabelecer potenciais correlações genotípicas-fenotípicas, tanto no que respeita à estrutura retiniana como às alterações funcionais. Métodos: 28 doentes de 26 famílias não relacionadas (12 homens, 16 mulheres) foram caracterizados do ponto de vista clínico, electrofisiológico (ERG), análise de mutações, tomografia de coerência óptica (OCT), autofluorescência, ressonância magnética nuclear craniana e testes de função renal. Resultados: O diagnóstico de Amaurose Congénita de Leber foi demonstrado em todos os doentes. A consanguinidade foi documentada em 25% das famílias. Clinicamente os doentes apresentavam-se com nictalopia em 21% dos casos e com o típico sinal oculo digital de Franceschetti em apenas 18% dos casos. A hiperopia foi o erro refractivo mais prevalente. Neste estudo, a aparência do fundo ocular variou entre o anatomicamente normal (4%), sem alterações específicas/atrofia do epitélio pigmentado da retina (25%), alterações pigmentares na periferia (64%) e defeitos maculares coloboma-like (25%). Observámos 29% dos casos com algum grau de atraso de desenvolvimento e 21% com sinais claros de autismo/comportamento autista. Apesar de a análise genética ainda estar em curso, até agora foram identificadas mutações causais em 5 doentes, dos quais 3 se 8 localizam no gene NPHP6, 1 no gene NPHP5 e outra no RPGRIP1. O doente com NPHP5 mutado foi posteriormente reclassificado como Síndroma de Senior-Loken. Os nossos resultados são compatíveis com os encontrados na literatura internacional. Conclusão: Caracterizamos, de um ponto de vista clínico e genético, a maior série de doentes Portugueses com LCA. O conhecimento aprofundado sobre esta condição é imprescindível para o aconselhamento e possível tratamento destes doentes, num futuro próximo

Conjunctival melanoma: association of cyclooxygenase-2 tumor expression to prognosis

PURPOSE: Conjunctival melanoma is a rare but potentially lethal tumor. Its biologic profile is still largely unknown, with recent studies aiming at establishing histopathological and genetic tumor profiles. The aim of this study was to analyze the association between clinicopathological characteristics and tumor expression of cyclooxygenase-2 (COX-2) to prognosis, assessing its usefulness as a possible prognostic marker. METHODS: Case series of 50 patients from 1991 to 2008 with pathologically proven conjunctival melanoma. Demographic, clinical, and pathological characteristics were evaluated by reviewing clinical files and pathology. Expression of COX-2 was studied by immunohistochemistry of formalin-fixed paraffin-embedded tissue samples of 20 melanomas. Samples were classified in a score which included intensity of staining and percentage of cells with positive reactivity. RESULTS: Clinicopathological features significantly associated (p < .05) with a poor prognosis (death) included involvement of fornix and tarsal conjunctiva, tumor thickness exceeding 2 mm, local tumor recurrence, lymph node, and systemic metastasis. In the immunohistochemistry study (n = 20), 18 cases expressed COX-2 although with different scores. However, only cases with a high score were associated with a poor outcome. Multivariate association analysis revealed that recurrence rate, metastasis, corneal invasion, and tumor thickness were associated with high score cases and, therefore, with a clinical profile with a higher risk of death. CONCLUSIONS: Results suggest that higher COX-2 expression may be a negative prognostic factor in conjunctival melanoma. Further studies can address the potential use of anti-COX-2 drugs as adjuvant therapy of this disease

Non-Hodgkin lymphoma with relapses in the lacrimal glands

Objective: To report an unusual case of systemic non-Hodgkin lymphoma (NHL) with repeated relapse in the lacrimal glands, in spite of complete remission for several years after treatment.Methods: A 78-year-old male with small lymphocytic B cell NHL, stage IV disease (lung invasion), was submitted to surgery and chemotherapy in 2001, with complete remission of the disease. In 2003 he developed a nodular lesion in the right lacrimal fossa. Pathology results revealed a local relapse of NHL. Radiation and chemotherapy were initiated and complete remission was again achieved. In 2012 the patient developed a new nodular lesion located in the left lacrimal fossa, resulting in diplopia, ptosis and proptosis of the left eye. Orbital computerized tomography (CT), ocular ultrasound and incisional biopsy were performed.Results: Orbital CT revealed a lesion infiltrating the left lacrimal gland and encircling the globe. Biopsy results confirmed a local relapse of B cell NHL. The patient was submitted to local radiation therapy with progressive resolution of ptosis, proptosis and diplopia. Response to treatment was monitored with ocular ultrasound. Conclusions: Patients with NHL diagnosis should be immediately investigated if ophthalmic or orbital symptoms develop. NHL extension to the orbit and adnexa is infrequent (5% of NHL cases) but may occur at any stage of the disease, including as a relapse site. In such cases, radiation and chemotherapy achieve good results, inducing long periods of remission

Rho kinase inhibitors - a review on the physiology and clinical use in Ophthalmology

© 2020 Springer Nature Switzerland AG. Part of Springer Nature.The Rho kinase (ROCK) signaling pathway is involved in several cellular events that include cell proliferation and cytoskeleton modulation leading to cell adhesion. The ROCK pathway in the human eye has been hypothesized to play important roles in corneal endothelial cell physiology and pathologic states. In addition, ROCK signaling has been identified as an important regulator of trabecular meshwork (TM) outflow, which is altered in glaucomatous eyes. These roles in corneal and glaucomatous disease states have led to the growing interest in the development of drugs selectively targeting this pathway (ROCK inhibitors). The authors provide a review of the literature on the pathobiology of the ROCK signaling in corneal endothelial disease, glaucoma, and vitreoretinal disease, as well as the clinical usefulness of ROCK inhibitors in Ophthalmology.info:eu-repo/semantics/publishedVersio