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

Summary

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.

Abstract

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality "omics" 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.

Introduction

Prostate cancer (PC) is the 2^nd most frequent cancer in men and the 5^th 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 disease^6,7. 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'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 metabolomics^13,14. 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'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 requirements^8,15,16.

Protocol

This method was developed through collaborative efforts involving different clinical services (Urology, Pathology, and the La Fe Hospital Biobank). The study was conducted in compliance with institutional, national, and international guidelines for human welfare, and it received approval from the Ethics Committee for Biomedical Research at the Instituto de Investigación Sanitaria Hospital Universitario y Politécnico La Fe (Valencia, Spain). All samples were stored at the La Fe Hospital Biobank (PT13/0010/0026). The overall procedure is detailed in Figure 1.

Figure 1: Overall procedure scheme. Schematic representation of the stepwise procedure described in the protocol section. Please click here to view a larger version of this figure.

1. Tumor targeting

1. Before surgery, utilize multiparametric or biparametric Magnetic Resonance Imaging (mpMRI or bpMRI)^17,18 to locate the tumor and map its position within the prostate gland. Combine the information obtained from MRI with findings from transrectal ultrasound or rectal examination if the latter yields discordant results.
2. To enhance tumor localization, review the results of preoperative ultrasound-guided and MRI-guided transrectal prostate biopsies.
   ​NOTE: In the diagnostic procedure for prostate cancer, biopsies are taken directly from the prostate via transrectal access. These biopsies include systematic sampling from the prostate (involving cylinders distributed all around the gland) and cylinders directly targeted to suspicious findings on MRI. To target the retrieval of these samples, transrectal ultrasound is used, which is combined with MRI findings in a cognitive manner ("cognitive fusion") or with "real MRI-ultrasound fusion software" when available.

2. Radical prostatectomy and prostate collection

1. Perform a standardized robotic-assisted radical prostatectomy through a transperitoneal approach, following the steps outlined by Huynh et al.¹⁹.
   1. Start with abdominal access, insufflation, and camera port placement (see Table of Materials). Place the remaining five ports under direct vision. Set pneumoperitoneum to 12 mm.
   2. Release the bladder by incising the peritoneum and dissect the perirectal space.
   3. Open the pelvic fascia on each side.
   4. Perform a complete dissection of the anterior and lateral aspects of the prostate and bladder.
   5. Carry out the opening of the bladder neck, identify and section the proximal urethra to gain access to the posterior aspects of the prostate and bladder.
   6. Dissect the seminal vesicles and rectum space¹⁹.
   7. Dissect the lateral aspects of the prostate, sparing the neurovascular bundles.
   8. Ensure hemostasis of vessels in the retropubic space.
   9. Dissect the apex and section the distal urethra.
   10. Complete the vesicourethral anastomosis.
   11. Extract the prostate and perform the closure of the incision¹⁹.
2. Collect the fresh prostate in a dry container. It is crucial not to fix or paraffin-embed the sample specimen.
3. Transport the fresh surgical specimen, which includes the entire prostate, to the Biobank for sample processing.

3. Prostate processing and sample collection

1. Select targeted prostate zones for sampling based on tumor location information retrieved from preoperative mpMRI, preoperative biopsy results, and direct prostatectomy specimen palpation. Record this information on a form that includes patient identification data, prostate areas finally sampled, and the number of samples collected.
   NOTE: When MRI findings, preoperative biopsy results, and direct prostatectomy specimen palpation are concordant, target all the samples to that or those locations (between 4 and 6 cylinders). If there is any discordance, increase the number of cylinders and separately target all zones that are suspicious of being affected by any of those methods.
2. Use a disposable automatic needle (surgeon) to retrieve cylindrical samples from the selected areas (step 1.1). Then, identify (pathologist) the outer zone of the cylinder, clamp the cylinder with tissue forceps, place it on a slide, and mark the outer zone with tissue ink. Repeat this process with all cylinders taken.
3. Place the collected cylinders horizontally on a sample holder disk (see Table of Materials) filled with cryostat embedding medium and transfer them to the microtome cooling platen for cutting.
4. Once placed on the platen, attach the sample holder disk to the specimen chuck, and cut it with careful movements of the wheel following the next steps:
   1. Use the optimal cutting temperature compound (OCT compound, see Table of Materials) to embed the tissue sample before frozen sectioning on a microtome-cryostat.
   2. Afterward, cut the sample using a cryostat-microtome at -25 °C. Collect thin cylinder-shaped sections of tissue (10 µm) onto treated microscope slides. Ensure to collect only a few microns of tissue to ensure an adequate amount of intact tumor tissue for storage in the Biobank.
   3. Gather two or three thin sections per slide and manually stain them to control the final coloration. The technician's eye ensures optimal staining, and any necessary re-staining can be done. Follow these steps
      1. Fix slides in 96% ethanol, hydrate in distilled water, and stain with Harris hematoxylin (see Table of Materials) for 1 min to stain the nuclei. Remove excess hematoxylin by rinsing the slides with distilled water.
      2. Treat slides with 30% ammonium hydroxide to turn the hematoxylin blue. Remove excess solution with rinses of distilled water.
      3. Stain slides with eosin (see Table of Materials) for 30 s.
      4. Next, dehydrate the slides with increasing titrations of alcohol, clear with xylene (100%), and mount them with DPX (a mixture of distyrene, a plasticizer, and xylene, used as a synthetic resin mounting media that replaces xylene-balsam, see Table of Materials). During these steps, dip the slides into and remove them from the jar of Coplin solution.
      5. Finally, place a coverslip on top of the stained tissue and use a medium with a suitable refractive index (see Table of Materials) for viewing under a microscope.

4. Sample characterization

1. Analyze the sample under a light microscope (pathologist), discriminating the areas of carcinoma and determining the Gleason score of the tumor. Document all these data and mark the precise tumor zones on the slides.
2. During this step, label the slide with the precise location of the tumor and its respective Gleason Score²⁰. Also, mark the location of PIN (Prostatic Intraepithelial Neoplasm) based on morphological identification.

5. Sample micro-macro-dissection and storage

1. Clearly mark different histological components with distinct markers on the slide (pathologist) and directly correlate them with frozen tissue on the disk.
2. Then, using a surgical blade, macro-micro dissect the areas of the sample marked on the slide within the OCT disc, dividing them into tumoral, peri-tumoral, PIN, and normal prostatic tissue.
3. As the embedding medium begins to defrost, handle the tissue with forceps and place it into correctly labeled cryotubes.
   NOTE: Carefully store each dissected fragment within the corresponding tube, following the histological diagnosis of the cryo-slices.
4. Store the labeled cryotubes with fully characterized histological samples in the Biobank at -80 °C for subsequent use in research studies.
   NOTE: The steps described in this section can be performed by a trained technician under the supervision of the pathologist. The surgical specimen should be transported to the Biobank as soon as possible to prevent tissue and nucleic acid degradation. According to the reference guides, efforts must be made to avoid prolonged ischemia time (>30 min)²¹. The samples can be kept in the Biobank indefinitely as long as the sample quality principles are maintained.

Results

The results reveal that this technique has made it possible to obtain tumor material in 61% of the cases studied (25 out of 41 cases) (Table 1).

Table 1: Histopathological data of study samples. Summary of histopathological data for the samples used in the study. The diagnostic cylinder refers to the prostate biopsy sample obtained for diagnostic purposes, while the processed cylinder corresponds to the cylinder obtained from the prostatectomy specimen for res...

Discussion

In any research study, obtaining quality samples is an essential requirement to reduce systematic biases and obtain reliable results²². Therefore, control of preanalytical variables such as the temperature at which samples are processed and stored, the time elapsed from sample collection to storage, the use of sterilized materials, or the effects that the addition of preservatives or other additives can have on samples must be considered in any protocol involving biological samples. Not only is th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cadiere forceps	Intuitive	PN1052082-US 10/2021	Part number: 471049. 18 uses.
Conventional slides	Knittel Glass	2021	Ground/ Frosted end
Cryostat microtome	Thermo Fisher Scientific	---	Criostato CryoStar NX50
Cryotubes	Greiner Bio-One GmbH	Ref.: 122280.	CRYO S. PP, with screw cap, sterile.
Da Vinci surgical system	Intuitive	PN1052082-US 10/2021	XI model
Dissection instruments	Bayer	---	Two tweezers and a surgical blade
DPX Eukitt	Medizin- und Labortechnik GmbH	6.00.01.0001.06.01.01
Eosin	Agilent	157252
Fenestrated bipolar forceps	Intuitive	PN1052082-US 10/2021	Part number: 471205. 14 lives.
Force bipolar	Intuitive	PN1052082-US 10/2021	Part number: 471405. 12 uses.
Freezers	Thermo Scientific		MODEL 907. -80 ºC
Hematoxylin	Agilent	157251
Inmunohistochemistry Slides	Agilent-Dako	K802021-2
Large needle driver	Intuitive	PN1052082-US 10/2021	Part number: 471006. 15 uses.
Maryland bipolar forceps	Intuitive	PN1052082-US 10/2021	Part number: 471172. 14 uses.
Microscope	Olympus	---	Olympus cx40
Microtome blades	PFM Medical	a35
Monopolar curved scissors	Intuitive	PN1052082-US 10/2021	Part number: 470179. 10 uses.
OCT compound	NEG-50	LOT.117340
PlusSpeed S Single-use Biopsy Device with beveled tip	Peter Pflugbeil GmbH	PSS-1825-S
ProGasp forceps	Intuitive	PN1052082-US 10/2021	Part number: 471093. 18 uses.
Sample holder Disc	Davidson Cryo Chuck. BradleyProducts		30 mm
Tissue ink	Pelikan	2021	Ink 4001 brilliant black (301168)
Xylol	Quimipur	Ref. 169