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.
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.
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'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'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.
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
2. Radical prostatectomy and prostate collection
3. Prostate processing and sample collection
4. Sample characterization
5. Sample micro-macro-dissection and storage
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 research purposes. ND: no data; LL: left lobule; RL: right lobule; TTB: total tumor burden. Please click here to download this Table.
For this analysis, the number of samples with the presence of tumor obtained in the cylinder acquired from the radical prostatectomy specimen from each of the lobules was compared with the presence of tumor in the cylinders of the biopsy performed for diagnosis. Specifically, the data reveal an efficiency of 59% (17/29) in the biopsies performed on the left lobe and 21% (7/33) in those performed on the right lobe (Table 1). Regarding the 16 prostates from which it was not possible to obtain a tumor during the sampling, 10 of them had a tumor load of less than 10%, and in no case did any of them present a tumor load of more than 20% (Table 1). A Fisher correlation test showed a capacity for obtaining statistically significant tissue when the tumor volume is at least 12% (p = 0.0187).
Finally, a receiver operating characteristic (ROC) curve analysis was carried out to determine the predictive performance of this method, obtaining an area under the curve (AUC) of 0.843 (Figure 2). These data indicate that this newly-developed method is capable of delivering satisfactory results with regard to tumor acquisition.
Figure 2: ROC curve. Receiver Operating Characteristic (ROC) curve illustrating the model's performance in binary classification of tissue samples as either tumor or non-tumor tissue. Please click here to view a larger version of this figure.
Lastly, to evaluate the concordance between the Gleason score of the tumor cylinder obtained by sampling and the Gleason score of the radical prostatectomy specimen, a Pearson's chi-squared analysis was performed. The results provided a concordance rate of 64%, increasing to 96% when immediate superior or nearest superior Gleasons were included (i.e., 3+ if 3+4; and 3+4 if 4+3). Figure 3 shows an example of the histopathological correlation of the radical prostatectomy specimen vs. the processed cylinder biopsy. It is important to note that all different histological patterns can be found in a single tissue sample: a tumor pattern with Gleason 3, tumor Gleason 4, PIN, and/or normal tissue.
Figure 3: Histopathological correlation of radical prostatectomy specimen vs. processed cylinder biopsy. (A) Hematoxylin and eosin (H&E) staining of needle core biopsy at 20x magnification, depicting Gleason pattern 3. Small, well-formed glands resembling normal prostate tissue are observed, lined by cancerous cells with distinctive features. (B) H&E staining of the specimen at 40x magnification, Gleason pattern 3. (C) H&E staining of needle core biopsy at 20x magnification, illustrating Gleason pattern 4. Irregularly shaped glands with enlarged, atypical cells are evident. (D) H&E staining of the specimen at 40x magnification, Gleason pattern 4. (E) H&E staining of needle core biopsy at 20x magnification, demonstrating Prostatic Intraepithelial Neoplasia (PIN). Crowded, overlapping cells within ductal spaces are observed. (F) H&E staining of needle core biopsy at 40x magnification, depicting PIN. (G) H&E staining of needle core biopsy at 20x magnification, representing normal prostate tissue. (H) H&E staining of needle core biopsy at 40x magnification, normal tissue. Scale bars = A,C,E,G,H, 0.5 mm; B,D,F, 0.05 mm. Please click here to view a larger version of this figure.
In conclusion, the most representative outcomes of the method presented herein include, on the one hand, the ability to obtain fresh intact samples (not fixed or paraffinized) characterized at the histological level to develop quality omics studies (genomic, transcriptomic, proteomic, metabolomic, among others). Additionally, it reduces bias associated with tumor heterogeneity in research studies, because each piece of micro- and macro-dissected tissue, stored in individual cryotubes, has a unique histology (Gleason, preneoplastic, normal tissue). The microscopic observation of the sample obtained makes it possible to define the different histological patterns found along the cylinder (Figure 3). Each tissue region with a given histological pattern is individually referenced, cut, and stored in a cryotube. Thus, each cryotube contains a tissue sample with unique histology. The analyses carried out on each of these samples will allow one to overcome the drawbacks of tumor heterogeneity. Other representative benefits include the reduced cold ischemia time in sample preprocessing. The average time from extraction to inclusion of the tissue sample in OCT was 13 min, and to storage, it was 33 min. This fact ensures better DNA and RNA quality. Finally, the establishment of fully characterized collections with information on clinical and pathological data, plus images of each of the samples obtained, guarded by a Biobank, can be used by the scientific community worldwide.
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 this key, but also the correct identification and codification of samples, as well as their pathological characterization, are crucial. Otherwise, there might be a large number of high-quality samples stored but remaining unused.
The collection and typification of tumor and non-tumor samples prior to storage in the Biobank are the main focus of this protocol due to their vast importance in the context of PC. Obtaining fresh PC tissue for research poses quite a challenge from a methodological point of view, due to the difficulty of successful tumor sampling9. Usually, targeted sampling in the prostate to collect tumor samples for research is not possible. Another limitation includes obtaining a representative tumor sample of the surgical specimen. The method presented here has successfully overcome these limitations, showing good performance in the acquisition of tumor samples, especially when the tumor burden exceeds 12% (which happens in most cases). Additionally, this method has provided a Gleason score correspondence between tissue cylinder and radical prostatectomy of 64%, rising to 96% when immediate superior or nearest superior Gleasons are included. These results are more than satisfactory, especially when compared to other studies previously performed for diagnostic purposes that show lower concordances23,24,25.
Moreover, compared to other published protocols, those with better results always require manipulation of the prostate specimen and a delay in the study. But correlating mpMRI, diagnostic biopsies, and manual inspection of the specimen prior to sample acquisition ensures less manipulation and better results than random sampling.
On the other hand, when a biopsy is collected, the tissue obtained can contain a heterogeneous population of cells with different histological characteristics, i.e., normal tissue, tumoral tissue with a low Gleason score, tumoral tissue with a high Gleason score, PIN, atypical small acinar proliferation (ASAP), etc. Consequently, the characterization and micro-macro dissection of this sample before carrying out the analysis allows for a more precise selection of the samples of interest and leads to more robust studies. Therefore, the greatest value of this methodological protocol lies within its ability to sample and characterize prostate tumor samples, ensuring high-quality samples and a complete report of their histological characteristics prior to storage. Although in this case all radical prostatectomies have been performed using the minimally invasive robotic surgical system, obtaining the sample using conventional techniques does not represent a problem. However, some critical steps have to be considered to obtain good results. On the one hand, control of the cold ischemia time between sample collection and processing, as this is directly related to DNA, RNA, and protein quality and integrity26. For this, the working team must be truly involved and implicated. On the other hand, it is crucial to have a pathologist or a technician with expertise in prostate cancer to typify, straightaway, the sample obtained to perform the macro-micro dissection of the sample.
Regarding the limitations of the method, the need to obtain a minimum amount of tissue from the prostate piece by means of targeted punctures guided by previous clinical data (i.e., MRI and biopsy data) can be highlighted. Another drawback is that this protocol can only be applied to a subgroup of prostate cancer patients, those undergoing radical prostatectomy. Finally, the use of OCT to embed tissue samples prior to frozen section on a microtome-cryostat may also be considered an impediment, especially in the analysis of data derived from metabolomic studies. However, this fact can be overcome by a series of washing steps, so the use of OCT is compatible with metabolomic studies as demonstrated in previous studies27,28,29.
In conclusion, the protocol presented here has potential applications in the field of prostate cancer research, but it can also serve as an insight for the collection and characterization of other types of human samples. Additionally, this protocol overcomes current limitations related to tumor heterogeneity, which has a great impact on the development of "omics" studies applied to biomedical research. These studies may further the identification of diagnostic, prognostic, or treatment response biomarkers, leading to the detection of subgroups of patients based on molecular characteristics.
The authors have nothing to disclose.
A.L. acknowledges a "Margarita Salas" postdoctoral contract (number 21-076), and MAM-T a 'Maria Zambrano' research contract (number MAZ/2021/03 UP2021-021). Both contracts have been funded by the European Union-Next generation EU.
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 |