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

Protocol for Prostatectomy Specimen Mapping and Dissection

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.

Author Spotlight: Advancing Prostate Cancer Research Through Improved Tissue Sampling and Biobanking

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

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.

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

To begin, use multi or biparametric MRI to locate the tumor and its position in the prostate gland before surgery. Next, prepare the patient with abdominal access, insufflation, and chemo port placement. Set the pneumoperitoneum to 12 millimeters.Make an incision on the peritoneum to release the bladder and dissect the perirectal space. Now, open the pelvic fascia on either side and perform a complete dissection of the anterior and lateral aspects of the prostate and bladder. Open the bladder neck and identify the proximal urethra.Section the proximal urethra to gain access to the posterior aspects of the prostate and bladder. Next, dissect the seminal vesicles and the rectum space, followed by the lateral aspects of the prostate. Dissect the apex.Then section the distal urethra. Then complete the vesico-urethral anastomosis. Extract the prostate, then suture the incision shut.Place the freshly collected prostate in a dry container. To process the prostate for sample collection, first select the targeted zones based on the MRI information. With a disposable automatic needle, retrieve cylindrical samples from the selected areas.Then identify the outer zone of the cylinder and clamp it with a pair of tissue forceps. Place the cylinder on a slide and mark the outer zone with tissue ink. Place the collected cylinders horizontally on a sample holder disc filled with cryostat embedding medium and transfer them to a microtome cooling plate for cutting.Next, attach the sample holder disc to the specimen chuck. Use the OCT compound to embed the tissue sample before sectioning. Use a cryostat microtome to cut the sample at 25 degrees Celsius.Collect thin cylinder-shaped tissue sections onto treated microscope slides. Fix the slides in 96%ethanol, then hydrate them in distilled water. Stain the sections in Harris hematoxylin for one minute.Now treat the slides with 30%ammonium hydroxide to turn the hematoxylin blue. Then stain the washed slides with eosin for 30 seconds. Then dehydrate the slides with increasing titrations of alcohol, followed by clearing with 100%xylene.Mount the slides with GPX and place a cover slip on the stained tissue. Use a light microscope to analyze the stained sample, discriminating areas with the carcinoma and determining the Gleason score of the tumor. Mark precise tumor zones on the slides.Then with the surgical blade, macro-microdissect the sample areas marked on the slide within the OCT disc. When the embedding tissue begins to defrost, lift the tissue with a pair of forceps and place them into correctly labeled cryotubes. This technique was used to obtain tumor material from 61%of the cases studied.Out of the 16 prostates were tumor acquisition was unsuccessful, 10 had less than 10%tumor load and none exceeded 20%An ROC curve analysis showed an area under the curve of 0.843, indicating that this method can deliver satisfactory tumor acquisition results. Histopathological correlation of the radical prostatectomy specimen versus the processed cylinder biopsy was performed using hematoxylin and eosin staining. Different histological patterns were found along the cylinder, such as Gleason pattern 3, which showed small well-formed glands lined by cancerous cells.Irregularly-shaped glands with enlarged atypical cells were observed in the Gleason pattern 4 sections. The samples with prostatic intraepithelial neoplasia showed crowded cells within ductal spaces.

radical-prostatectomy-prostate-staining-sample-characterization-for

Research

JoVE Journal

Cancer Research

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be particularly challenging in the context of prostate cancer (PC) due to the unique nature of this organ and the high heterogeneity associated with this tumor. On the other hand, histopathologically characterizing samples before their storage without causing significant tissue alterations is also an intriguing challenge. In this context, we present a new method for acquiring, mapping, characterizing, and micro-dissecting resected prostate tissue based on anatomopathological criteria.
Unlike previously published protocols, this method reduces the time required for histopathological analysis of the prostate specimen without compromising its structure, which is crucial for assessing surgical margins. Furthermore, it enables the delineation and micro-macro dissection of fresh prostate tissue samples, with a focus on histological tumor areas defined by pathological criteria such as Gleason score, precursor lesions (high-grade prostatic intraepithelial neoplasia - PIN), and inflammatory lesions (prostatitis). These samples are then stored in a Biobank for subsequent research analyses.

This protocol describes a method for facilitating the collection of samples from radical prostatectomy specimens. The goal is to map, characterize, and micro-macro dissect tissue samples from the specimens based on anatomopathological criteria before storing them in a Biobank.