Phenotype-Genotype Correlation in Colorectal Cancer: A Real-Life Study.

BACKGROUND AND AIMS: Colorectal cancer (CRC) is a heterogeneous disease with distinctive genetic pathways, such as chromosomal instability, microsatellite instability and methylator pathway. Our aim was to correlate clinical and genetic characteristics of CRC patients in order to understand clinical implications of tumour genotype. METHODS: Single-institution retrospective cohort of patients who underwent curative surgery for CRC, from 2012 to 2014. RAS and BRAF mutations were evaluated with the real-time PCR technique Idylla®. Mismatch repair deficiency (dMMR) was characterized by absence of MLH1, MSH6, MSH2 and/or PMS2 expression, evaluated by tissue microarrays. Overall survival (OS) and disease-free survival (DFS) were assessed using survival analysis. RESULTS: Overall, 242 patients were included (males 57.4%, age 69.3 ± 12.9 years; median follow-up 49 months). RAS-mutated tumours were associated with reduced DFS (p = 0.02) and OS (p = 0.045) in stage I-III CRC. BRAF-mutated tumours were more predominant in females and in the right colon, similarly to dMMR tumours. BRAF status did not influence OS (4 years)/DFS (3.5 years) in stage I-III disease. However, after relapse, length of survival was 3.5 months in BRAF-mutated tumours in contrast to 18.6 months in BRAF wild-type tumours (p = NS). No germline mutations in mismatch repair genes were so far identified in the patients with dMMR tumours. Molecular phenotype (RAS, BRAF and MMR) did not influence OS in metastatic patients. Our small sample size may be a limitation of the study. CONCLUSION: In our cohort, RAS-mutated tumours were associated with worse DFS and OS in early-stage CRC, whereas the remaining molecular variables had no prognostic influence.

INTRODUÇÃO: O cancro colo-rectal (CCR) é uma doença heterogénea, com vias genéticas distintas, nomeadamente instabilidade cromossómica, instabilidade de microssatélites e via metiladora. O nosso objetivo foi correlacionar as características clínicas e genéticas dos doentes com CCR e, deste modo, conhecer as implicações na prática clínica do genótipo tumoral. MÉTODOS: Estudo de coorte retrospectivo unicêntrico de doentes diagnosticados com CCR e submetidos a cirurgia com intuito curativo, entre 2012 e 2014. As mutações RAS e BRAF foram avaliadas pela técnica de real time PCR Idylla®. A deficiência de mismatch repair (MMR) foi avaliada pela técnica de tissue microarrays e definida pela ausência de expressão de MLH1, MSH6, MSH2 e/ou PMS2. A sobrevivência global (SG) e a sobrevivência livre de doença (SLD) foram avaliadas por análise de sobrevivência. RESULTADOS: No total, foram incluídos 242 doentes (homens 57.4%, idade 69.3 ± 12.9 anos, mediana de seguimento de 49 meses). Os tumores RAS-mutados associaram-se a menor SLD (p = 0.02) e SG (p = 0.045) em doentes com CCR estadio I­III. Os tumores BRAF-mutados foram mais frequentes em mulheres e nos tumores do cólon direito, assim como os tumores com deficiência para MMR. O status BRAF não influenciou a SG (4 anos)/SLD (3.5 anos) nos estadio I­III. Contudo, após a recidiva, o tempo de sobrevivência foi de 3.5 meses nos tumores BRAF-mutados, em comparação com 18.6 meses nos tumores sem esta mutação (p = NS). Não se identificaram mutações germinativas nos genes de mismatch repair nos doentes com tumores deficientes para estas proteinas (dMMR). O perfil molecular (RAS, BRAF e MMR) não influenciou a sobrevivência global dos doentes com metástases ao diagnóstico. O tamanho da amostra pode ser uma limitação do estudo. CONCLUSÃO: Na nossa coorte, os tumores RASmutados associaram-se a pior SLD e SG nos estádios precoces de CCR. Os restantes marcadores moleculares não influenciaram o prognóstico dos doentes.

Primary ciliary dyskinesia due to CCNO mutations-A genotype-phenotype correlation contribution.

Primary ciliary dyskinesia (PCD) is genetically and clinically heterogeneous. CCNO mutations are associated with chronic destructive lung disease and were first described in 2014. Early reports suggest that CCNO is mutated more frequently than expected, however, these are considered rare. We report on three eleven-year-old children with PCD due to CCNO mutations. All children presented early-onset respiratory symptoms, no cardiac or situs anomalies and moderate to severe clinical courses. Patients 1 and 3 were admitted to a neonatal intensive care unit due to respiratory distress. Patients 1 and 2 had atelectasis and lobar collapse, for which lobectomy was performed for patient 1. Patient 3 also presented otitis media with effusion with conductive hearing loss, requiring tympanostomy tube insertion twice. Diagnosis of PCD for all three required repeated nasal brushings, delaying diagnostic confirmation. Microscopy analysis revealed severely decreased numbers of cilia, but normal ultrastructure and uncoordinated beat pattern in the residual cilia. Surprisingly, the prevalence of pathogenic CCNO variants in our centre is higher than expected (three out of sixteen patients). Pathogenic variants in PCD-causing genes lead to specific ultrastructural defects, and there is a suggestion for genotype-phenotype association. However, there are little longitudinal data evaluating the impact of specific defects on disease progression, but a recent study showed a worse lung disease and poorer nutritional status. Concluding, this report underlies the importance of patient-oriented diagnosis and management in highly experienced PCD centres.

Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue.

BACKGROUND: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. METHODS: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. RESULTS: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. CONCLUSIONS: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.

Farnesoid X Receptor Expression in Microscopic Colitis: A Potential Role in Disease Etiopathogenesis.

INTRODUCTION: Microscopic colitis (MC) is a chronic inflammatory bowel disease with unclear etiology. Bile acid (BA) malabsorption has been described in MC patients. Farnesoid X receptor (FXR) is the main BA receptor; FXR-mediated mechanisms prevent the noxious effects of BA accumulation, preserving the integrity of the intestinal epithelial barrier and preventing intestinal inflammation. AIM: Our aim was to describe the expression of FXR in patients with MC. METHODS: Archival formalin-fixed paraffin-embedded samples from the terminal ileum, right and left colon were obtained from patients with MC and matched controls. Immunohistochemistry was performed and nuclear FXR expression scored in a semi-quantitative way. RESULTS: 169 formalin-fixed paraffin-embedded samples from 35 patients with MC and 31 controls were retrieved. There was a significant reduction of FXR expression in patients with MC versus controls both in the right colon (moderate-strong FXR expression: 21.1 vs. 64.3%; p = 0.003) and left colon (moderate-strong FXR expression: 8.3 vs. 38.7%; p = 0.027). No significant differences in FXR expression were observed in the ileum of patients with MC (moderate-strong FXR expression: 76.9 vs. 90.9%; p = 0.5). We found no difference in FXR expression between the two types of MC. No association between the degree of lymphocyte infiltration or the thickness of collagen band and FXR expression was found. CONCLUSIONS: Patients with MC present a significantly lower expression of FXR in the colon. This could render colonic epithelial cells more susceptible to the deleterious effects of BA, contributing to disease pathogenesis and symptoms in MC.

INTRODUÇÃO: A colite microscópica (CM) é uma doença inflamatória do intestino com etiologia desconhecida. A má-absorção de ácidos biliares (AB) encontra-se descrita em doentes com CM. O Recetor Farnesoid X (FXR) é o principal recetor dos AB; mecanismos mediados pelo FXR previnem o efeito nocivo da acumulação dos AB, preservando a integridade da barreira epitelial intestinal e impedindo inflamação. OBJETIVOS: Avaliar a expressão do FXR em doentes com CM. MÉTODOS: Foram obtidas amostras em parafina do ileon terminal, cólon direito e esquerdo de doentes com CM e controlos emparelhados; foi realizada imunohistoquímica para FXR e a sua expressão nuclear quantificada de forma semi-quantitativa. RESULTADOS: Foram estudadas 169 amostras de 35 doentes com CM e 31 controlos. A expressão de FXR nos doentes com CM foi significativamente inferior à dos controlos no cólon direito (expressão FXR moderada-intensa: 21.1% versus 64.3%; p = 0.003) e esquerdo (expressão FXR moderada-intensa: 8.3% versus 38.7%; p = 0.027). Não observámos diferenças significativas na expressão do FXR no ileon em doentes com CM em comparação com controlos (expressão moderada-intensa em 76.9 versus 90.0%; p = 0.5). Não se verificaram diferenças na expressão FXR entre os dois tipos de CM. Não se encontrou associação entre o grau de infiltração linfocítica ou a espessura da banda de colagénio e a expressão de FXR. CONCLUSÕES: os doentes com CM apresentaram uma expressão significativamente reduzida do FXR no cólon; isto pode tornar as células epiteliais mais suscetíveis aos efeitos deletérios dos AB, contribuindo para a patogénese da doença e a sintomatologia da CM.