radical prostatectomy, prostate staining, and sample characterization for tumor identification

Protocolo para Mapeamento e Dissecção de Espécimes de Prostatectomia

Prostate cancer (PC) is the 2nd most frequent cancer in men and the 5th leading cause of death worldwide1. Patient treatment and prognosis depend on the staging and grading (Gleason score) of the tumor, as evidenced by the higher 5-year survival rates of localized and low-grade tumors (Gleason grade 6) (99%) compared to high Gleason grades and metastatic tumors (31%)2.
PC local relapse and treatment failure have been linked to the characteristic high genetic intratumor heterogeneity of this tumor type3. Additionally, PC is considered to be a multifocal disease with several tumor foci exhibiting different morphological, histological, and molecular characteristics4, which may originate independently or derive from a common tumor cell ancestor5. Previous studies have shown that tumor evolution differs among patients based on specific genetic drivers that can promote metastasis or confine the cell lineage to the prostate5. Therefore, molecular characterization of the different tumor foci is crucial not only for providing a more accurate diagnosis and prognosis but also for tailoring effective and personalized treatment for the patient.
In this context, biomedical research and integrative multi-omics approaches are offering unprecedented opportunities to classify cancers into different subtypes, identify diagnostic and prognostic biomarkers, and discover markers related to treatment response. Furthermore, these approaches contribute to a better understanding of the biology of this disease6,7. Biological samples, whether tissues or biofluids, can be analyzed using various multi-omics platforms (genomics, transcriptomics, proteomics, metabolomics, etc.) to uncover the biological features underlying cancer pathophysiology, thereby addressing current limitations related to genetic and phenotypic heterogeneity6. However, it&#39;s important to consider that the quality of data derived from omics studies depends on the quality of the samples collected from tumors, their accurate characterization, and subsequent processing and storage8.
In this context, obtaining fresh PC tissue for research presents a methodological challenge due to the difficulty of successful tumor sampling9. Previous methods involved random sampling following radical prostatectomy, yielding poor results10. However, more recent approaches incorporate targeted protocols based on both magnetic resonance imaging (MRI) and biopsy data, resulting in improved efficacy in tumor sample collection11.
On the other hand, histopathological characterization of samples prior to their storage without significant tissue alteration also poses an interesting challenge. Consequently, in many cases, the histopathological determination of samples is performed after their analysis (e.g., HR 1H NMR metabolomic analysis)12. This practice entails unnecessary expenses, time consumption, and the loss of a significant number of samples that are eventually excluded from the analysis (for example, samples that, following histopathological analysis, turn out not to be tumor samples). In other cases, the histopathological characterization of samples is performed before their analysis. In fact, some previous studies have attempted to standardize methods for providing representative high-quality research samples from radical prostatectomy specimens for genomics and metabolomics13,14. Nevertheless, sampling efficiency is significantly higher when performed from already histologically confirmed sections (88%) that disrupt tissue, compared to when performed from unconfirmed sections (45%)1.
Here, a new methodology is presented to overcome these limitations, aiming to obtain fresh and well-characterized PC samples before storage in the Biobank. This method has been developed through collaborative efforts between different clinical services (Urology, Pathology, and the La Fe Hospital Biobank). It&#39;s important to highlight that Biobanks play an essential role in the collection, processing, preservation, and storage of biological samples while ensuring the high quality of samples and data, as well as compliance with ethical and legal requirements8,15,16.

Autor em destaque: Avançando na pesquisa do câncer de próstata por meio de amostragem de tecido aprimorada e biobanco

Melhorando a confiabilidade do biobanco de tumores de próstata com técnica de amostragem aprimorada e caracterização histológica

Our research focuses on enhancing the methodology to obtain high quality prostate cancer tissue samples. It addresses challenges like genetic intratumoral heterogeneity, multifocality, and precise molecular characterization of tumor foci in prostate cancer research. Biobanking is a technology driving practice that systematically collects processes and stores biological samples.Integrating these technologies enables the generation of extensive dataset from well characterized prostate cancer samples, fostering collaboration, improving research, reliability, and aiding in the development of personalized medicine and strategies for prostate cancer patients. Pathology faces several experimental changes including intratumoral heterogeneity, histopathological standardization, molecular data integration, lab intensive processes, climate quantitative analysis underneath for automated e-image analysis. Addressing these issues requires ongoing technological advancements, a standardization initiatives, interdisciplinary collaborations, and improved experimental and computational methods.The protocol handles tumor sampling, efficiently and accurately. Integrates imaging and biopsy data for precise localization. Offers realtime histopathological insights, reduces costs and time, improves sample quality, and minimize all ischemia time.This addresses critical challenges in prostate cancer histopathological research. Our protocol can transform prostate cancer research by improving tumor understanding both histopathological data, enhancing biobanking, fostering collaboration, and enabling personalized medicine. Longitudinal biobank reveals temporal changes, clarifying disease progression and treatment responses, interacting multi data, uncovers molecular nerves and novel biomarkers for prostate cancer.

Prostatectomia Radical, Coloração da Próstata e Caracterização de Amostras para Identificação de Tumores

Para começar, use ressonância magnética multi ou biparamétrica para localizar o tumor e sua posição na próstata antes da cirurgia. Em seguida, prepare o paciente com acesso abdominal, insuflação e colocação de porta de quimioterapia. Defina o pneumoperitônio para 12 milímetros.Faça uma incisão no peritônio para liberar a bexiga e dissecar o espaço perirretal. Agora, abra a fáscia pélvica de ambos os lados e faça uma dissecção completa dos aspectos anterior e lateral da próstata e da bexiga. Abra o colo da bexiga e identifique a uretra proximal.Corte a uretra proximal para obter acesso aos aspectos posteriores da próstata e da bexiga. Em seguida, disseque as vesículas seminais e o espaço reto, seguidos pelos aspectos laterais da próstata. Disseque o ápice.Em seguida, corte a uretra distal. Em seguida, complete a anastomose vesico-uretral. Extraia a próstata e suture a incisão.Coloque a próstata recém-coletada em um recipiente seco. Para processar a próstata para coleta de amostras, primeiro selecione as zonas-alvo com base nas informações de ressonância magnética. Com uma agulha automática descartável, recupere amostras cilíndricas das áreas selecionadas.Em seguida, identifique a zona externa do cilindro e prenda-o com uma pinça de tecido. Coloque o cilindro em uma lâmina e marque a zona externa com tinta de tecido. Coloque os cilindros coletados horizontalmente em um disco porta-amostras cheio de meio de incorporação de criostato e transfira-os para uma placa de resfriamento de micrótomo para corte.Em seguida, prenda o disco porta-amostras ao mandril da amostra. Use o composto OCT para incorporar a amostra de tecido antes do corte. Use um micrótomo criostato para cortar a amostra a 25 graus Celsius.Colete seções finas de tecido em forma de cilindro em lâminas de microscópio tratadas. Fixe as lâminas em etanol a 96% e hidrate-as em água destilada. Manchar as seções em hematoxilina de Harris por um minuto.Agora trate as lâminas com hidróxido de amônio a 30% para tornar a hematoxilina azul. Em seguida, manche as lâminas lavadas com eosina por 30 segundos. Em seguida, desidrate as lâminas com titulações crescentes de álcool, seguidas de limpeza com 100% de xileno.Monte as lâminas com GPX e coloque uma lamínula no tecido manchado. Use um microscópio óptico para analisar a amostra corada, discriminando áreas com o carcinoma e determinando o escore de Gleason do tumor. Marque zonas tumorais precisas nas lâminas.Em seguida, com a lâmina cirúrgica, macromicrodissecar as áreas de amostra marcadas na lâmina dentro do disco OCT. Quando o tecido de incorporação começar a descongelar, levante o tecido com uma pinça e coloque-os em criotubos corretamente rotulados. Essa técnica foi utilizada para obter material tumoral de 61% dos casos estudados.Das 16 próstatas em que a aquisição do tumor não foi bem-sucedida, 10 tinham menos de 10% de carga tumoral e nenhuma excedeu 20% Uma análise da curva ROC mostrou uma área sob a curva de 0,843, indicando que este método pode fornecer resultados satisfatórios de aquisição do tumor. A correlação histopatológica da peça de prostatectomia radical versus a biópsia do cilindro processada foi realizada usando coloração de hematoxilina e eosina. Diferentes padrões histológicos foram encontrados ao longo do cilindro, como o padrão de Gleason 3, que mostrou pequenas glândulas bem formadas revestidas por células cancerígenas.Glândulas de formato irregular com células atípicas aumentadas foram observadas nas seções do padrão 4 de Gleason. As amostras com neoplasia intraepitelial prostática mostraram células apinhadas dentro dos espaços ductais.

Research

JoVE Journal

Cancer Research

Radical Prostatectomy, Prostate Staining, and Sample Characterization for Tumor Identification

Enhancing Prostate Tumor Biobanking Reliability with Improved Sampling Technique and Histological Characterization

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be ...

Pesquisa do câncer