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Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy
EUF-655; No. of Pages 18 E U R O P E A N U R O L O GY F O C U S X X X ( 2 0 18 ) X X X– X X X
available at www.sciencedirect.com journal homepage: www.europeanurology.com/eufocus
Prostate Cancer
Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy Daniele Panarello a,b, Eva Compe´rat c, Olivia Seyde c, Alexandre Colau a, Carlo Terrone b, Bertrand Guillonneau a,* a
Service of Urology, Diaconesses—Croix St Simon Hospital, Sorbonne University, Paris, France;
b
Department of Urology, San Martino Hospital, Genoa
University, Genova, Italy; c Service of Pathology, Tenon Hospital, HUEP, Sorbonne University, Paris, France
Article info
Abstract
Article history: Accepted January 8, 2019
Background: Confocal laser endomicroscopy (CLE) is an optical device that aims to image histological architecture and may be used to reduce positive surgical margins. The ability of CLE to describe prostatic and periprostatic tissues, and prostate cancer (PCa) is still an object of investigation. Objective: To create an atlas of ex vivo CLE images of prostatic and periprostatic tissues, and PCa in order to recognise different prostatic structures. Design, setting, and participants: From November 2017 to February 2018, 15 patients underwent radical prostatectomy for biopsy-proven PCa. Outcome measurements and statistical analysis: Based on preoperative data and macroscopic examination, tumour location was assessed and confirmed on frozen sections. Prior to ex vivo CLE analysis, prostates were stained with fluorescein 10%. We used a GastroFlex probe to collect images of periprostatic tissue (adipose tissue, fibrous and connective tissues, vessels, nerve sheets, seminal vesicles, and urethra). Normal prostatic glands and tumour tissue according to the Gleason grade were analysed. Each PCa Gleason score was represented. Results and limitations: A total of 139 video clips and 237 pictures of prostatic and periprostatic tissues were collected. Among them, we selected 16 highly representative images. Adipose tissue, fibrous tissue, and connective tissue were supposable in all 15 specimens. PCa glands captured fluorescein in their cytoplasm, normal prostatic glands did not capture fluorescein, and glandular structures were easily recognisable. The principal limitation of this study is its ex vivo nature of the study. Conclusions: Each CLE image was correlated with the corresponding haematoxylin/ eosin/saffron definitive pathology image, allowing building of an atlas as a necessary tool to assess the diagnostic performance of CLE during radical prostatectomy in achieving negative surgical margins. Patient summary: In this study, we aim to provide an atlas of images illustrating prostatic, periprostatic, and PCa tissues obtained using Cellvizio confocal laser endomicroscopy as a tool for further interpretation of intraoperative surgical margins during radical prostatectomy. © 2019 Published by Elsevier B.V. on behalf of European Association of Urology.
Associate Gratzke
Editor:
Christian
Keywords: Confocal Laser Endomicroscopy Prostate Prostate Cancer
* Corresponding author. Service of Urology, Diaconnesses—Croix St Simon Hospital, 125 rue d’Avron, 75020, Paris, France. Tel.: +33 1 44 74 10 56. E-mail address: [email protected] (B. Guillonneau).
https://doi.org/10.1016/j.euf.2019.01.004 2405-4569/© 2019 Published by Elsevier B.V. on behalf of European Association of Urology.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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1.
Introduction
laparoscopic or robotic-assisted surgery was performed. No fluorescein was intravenously injected during surgery. All patients provided written
Prostate cancer (PCa) is the second most commonly diagnosed cancer in men. The incidence of PCa diagnosis in Western and Northern Europe is 94.9 and 85 per 100,000 age-standardised rate [1]. Different treatments are possible for PCa: for localised disease, the options are active surveillance, radical prostatectomy (RP), and radiation therapy. RP is an alternative to active surveillance for low-risk PCa patients who accept a trade-off between toxicity and prevention of disease progression; it is recommended in intermediate-risk patients and can be considered in high-risk PCa [1]. The goal of RP is complete removal of disease while preserving continence and potency whenever possible [2]. Following Walsh and Donker’s [3] initial study, preservation of neurovascular-bundles (NVBs) is mandatory to preserve potency and improve continence after RP. Despite the use of magnification in laparoscopic and robot-assisted RP, positive surgical margins (PSMs) continue to affect the oncological results. A recent literature review on the results of surgical techniques published between 2008 and 2011 reported a PSM prevalence ranging from 6.5% to 32%, with a mean value of 15% when not stratified by pathological stage. The overall mean PSM rate was 9% (range: 4–23%) in pT2, 37% (range: 29–50%) in pT3, and 50% (range: 40–75%) in pT4 cancers [4]. PSM status is correlated with biochemical recurrence (BCR) [5] and, even if still debated [6], could affect PCa-specific mortality [7]. In order to improve the rate of nerve-sparing surgery while reducing PSM rate, the Martini Klinik group published in 2012 their case series assessing the impact of the NeuroSAFE technique on PSM, BCR, and nerve sparing [8]. NeuroSAFE uses intraoperative frozen sections to determine whether a nerve-sparing approach is possible. Nowadays, many technologies aim to increase the visual capability and define cellular architecture in vivo. The confocal laser endomicroscopy (CLE) system Cellvizio (Manua Kea Technologies, Paris, France) is an optical device based on a 488-nm blue laser used in combination with fluorescein, providing high-resolution imaging of spatial cellular disposition similar to histopathology [9]. Presently, it is used for bladder, upper urinary tract [9], gastrointestinal tract [10], and lung [11] tumours. CLE was preliminary tested during robot-assisted RP, and tissues were sampled ex vivo and in vivo without extensive description of different tissues analysed [12]. The aim of our study was to create an atlas of ex vivo prostate tissue images using CLE, analysing periprostatic tissue, normal prostatic tissue, and PCa images to provide an illustrated guide useful for future intraoperative evaluation of surgical margin (SM) status during RP. The rational was to recognise tumour tissue in case of tumour-positive margins, and to differentiate from nontumoural intraprostatic margins. 2.
Patients and methods
From November 2017 to February 2018, we selected 15 patients eligible for RP after biopsy-proven PCa with different preoperative Gleason
informed consent. All prostate specimens were immediately preserved fresh within a tissue-safe vacuum-sealed package in the operative room, which was delivered to the pathology laboratory, where packages were unsealed when the study examination started. A senior genitourinary (GU) pathologist assessed the tumour location based on macroscopic examination, preoperative prostate magnetic resonance imaging (MRI) results, and positive biopsy topography, and then performed targeted frozen sections. The fresh frozen procedure was performed according to the pathology standards in order to confirm the presence of PCa, and the slices were stained by haematoxylin/eosin/saffron (HES). In order to recognise the examined areas on definitive histopathological samples, different coloured inks were used to stain the areas of interest. The staining protocol is shown in Table 1. We first focused on periprostatic tissue to identify the outer layer of the prostate (adipose, fibrous, and connective tissue); we then evaluated the structures of the outer layer such as vessels, nerve sheets, seminal vesicles, and urethra. Furthermore, we aimed to recognise normal prostatic glands and tumour glands. The Gleason score was used according to the World Health Organization (WHO) 2016 classification. We utilised a GastroFlex probe for imaging. The technical specifications are as follows: maximal field of view 240 mm, depth 55–65 mm,
lateral resolution 1 mm, and magnification comparable with 800 at optic microscopy. Regarding fluorescein-staining protocol, for the first two cases, we used the protocol previously published [12], which consisted of 2-min dipping in a solution of fluorescein and 7-min washing in saline solution. This was then modified for a protocol of 10 ml fluorescein 10% (100 mg/ ml) and 500 ml of saline solution. We dipped the prostate in this solution for 15 s followed by 60 s in water. Prostates examined after >12 h from surgery had poorer image quality and the fluorescein dipping was insufficient; the specimens were therefore immersed in the fluorescein solution for 60 s followed by 120 s of washout. Every sequence was video recorded, as well as the time of relevant images together with their spatial distribution. All videos were reviewed by the pathologist and the urologist. The images and findings were correlated with the final pathology. The correlation was made with the use of different coloured inks, which marked the areas where the videos were recorded. After the classical histopathology handling, we were able to find the exact location of the initial video, and superposition
was made easily.
Table 1 – Staining protocol.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time from surgery
Stain protocol
Number of pictures
Number of clips
12:00 14:00 12:00 1:00 12:00 12:00 1:00 1:00 14:00 12:00 1:00 14:00 12:00 16:00 1:00
2 + 7 min 2 + 7 min 60 + 120 s 15 + 60 s 60 + 120 s 60 + 120 s 15 + 60 s 15 + 60 s 60 + 120 s 60 + 120 s 15 + 60 s 60 + 120 s 60 + 120 s 60 + 120 s 15 + 60 s
7 10 18 14 34 16 17 17 8 9 11 16 14 26 20
2 3 5 13 14 6 6 9 6 7 13 8 12 17 18
scores. We analysed the prostate specimens using CLE ex vivo. Either
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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3.
Results
The final Gleason scores of PCa after standard histopathological techniques, used according to WHO 2016 recommendations [13], were as follows: one specimen with Gleason score 6 (3 + 3), five with Gleason score 7 (3 + 4), three with Gleason score 7 (4 + 3), four with Gleason score 8 (4 + 4), and one with Gleason score 9 (5 + 4) prostate adenocarcinoma. One patient, who
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presented a Gleason score of 9 (5 + 4) on the biopsy, was not graded due to neoadjuvant hormonal treatment leading to morphological alterations; nevertheless, the images were examined and recorded (Fig. 1). Patient characteristics are shown in Table 2. The delay between surgery and CLE imaging examination ranged from 1 to 18 h. Five prostate specimens were analysed within 1 h from surgery, and 10 specimens were
Fig. 1 – Periprostatic tissue. HES = haematoxylin/eosin/saffron.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Table 2 – Patient characteristics.
PSA prebiopsy Clinical stage
Biopsy Gleason score
Biopsy ISUP grade group
Gland volume Pathological stage
Pathological Gleason score
Pathological ISUP grade group
Max tumour diameter Number of video clips per specimen Number of pictures per specimen
Mean
Median
9.75
7.36
cT1c cT2a cT2b cT2c cT3a 6 7 9 1 2 3 5 42.9
%
3 6 5 0 1 3 10 2 3 8 2 2
20.0 40.0 33.3 0.0 6.7 20.0 66.7 13.3 20.0 53.3 13.3 13.3
6 4 5 1 8 4 1 1 5 3 4 1
7.1 57.1 28.6 7.1 7.1 35.7 21.4 28.6 7.1
42.5
pT2 pT3a pT3b 6 7 8 9 1 2 3 4 5 23 9 16
n
20 8 16
ISUP = International Society of Urological Pathology; PSA = prostatespecific antigen.
analysed at >12 h (range 1—18 h). All frozen section slices were positive for PCa. We collected 139 different video clips (mean: nine clips per specimen) and 237 pictures (median: 16 pictures per specimen) of prostatic and periprostatic tissues. The mean time of video recording per patient was 8.53 min (range 2–29 min). Among the recorded sequences, we identified 237 pictures relevant for the CLE PCa atlas. Among them, we selected 52 informative images illustrating the different aspects of prostate tissue and PCa (Figs. 1–13). Among them, we superselected 16 very informative images of periprostatic tissues, prostatic tissues, and PCa (Figs. 1–4). The remaining 36 images (Figs. 5–13) were included in the atlas. At first, the outer layers of all 15 prostate specimens were examined. Adipose tissue, fibrous tissue, and connective tissue were comparable in all 15 specimens. Vessels and nerves were more difficult to distinguish from each other, but structures corresponding to nerve sheets and vessels were found and reported. Then, we examined the prostate apex and the surrounding muscular and urethral tissues, which displayed an excellent visual correlation with HES staining. Both cellularity and spatial distribution were visually comparable with the final pathology (Figs. 2 and 5 –7). Once the pathologist was trained in working with high magnification, glandular nontumoural intraprostatic tissues became easily recognisable (Figs. 3, 8, and 9). Periprostatic tissues were recognised and distinguished in CLE images since their histological architecture was perfectly comparable with the architecture of HES staining.
Adipocytes appear like large, black, round, nonfluorescent uniform cells with clear membranes (Figs. 2, 5, and 6). Smooth muscle is composed of multiple “cigar”-shaped cells (Figs. 2 and 5 –7); connective tissue surrounding the prostate is recognisable by the shape of the fibres within the extracellular matrix (Figs. 2 and 5 –7). Prostatic apex is composed of muscular cells, connective tissue, and rare glands. Nerves were recognisable especially in longitudinal sections. Regarding seminal vesicles, irregularity of the glandular lumen together with the presence of smooth muscle cells guided the interpretation of images. Basal cell hyperplasia was also observed (Fig. 3). These cells never displayed any fluorescence; nevertheless, they allowed us to differentiate between intra- and extraprostatic tissues. We were able to distinguish normal prostate glands from tumour tissue by fluorescence: PCa glands were identified for their early ability in capturing fluorescein in their cytoplasm, while normal prostatic glands did not capture fluorescein (Figs. 1, 3, 4, 8 –13). Regarding the various Gleason grades, we found either well-defined glands with cytoplasmic fluorescein emission corresponding to well-differentiated PCa of Gleason grade 3 or less well-differentiated aspects. The most easily recognisable Gleason grade was Gleason grade 4, especially glomeruloid and confluent glands with rigid patterns. Intraglandular tumour cells were easily seen, and some had papillae-like propulsions. The patient treated with neoadjuvant hormonotherapy also displayed easily recognisable aspects: the glands were of bigger size with a slightly fluorescent content, with the surrounding tumour glands capturing fluorescein as well. The most difficult correlation was related to poorly differentiated tumour glands; nevertheless, the latter showed some emission of fluorescein as well. 4.
Discussion
Images of prostatic and periprostatic tissues provide good CLE images to be used by pathologists and surgeons when starting their experience with CLE during RP. Presently, the decision to perform nerve-sparing surgery, either complete or partial, uni- or bilaterally, or to perform full resection of the neurovascular bundle is mainly based upon preoperative clinical features: prostate-specific antigen test, clinical stage, biopsy results, and prostate MRI analysis [1]. We do not yet have tools to assess the SM status intraoperatively; according to the European Association of Urology guidelines, intraoperative frozen section can be helpful in making the decision of whether to perform a nerve-sparing surgery [1]; nevertheless, this can lead to a less accurate definitive pathology analysis. For this reason, there is a rationale for tools on surgeon guidance during NVB dissection, ensuring first of all negative SMs and the optimal nerve-sparing approach while optimising oncological safety. We identified an improved protocol for ex vivo imaging based on a staining process in a fluorescein 0.2% solution. To date, only Lopez et al [12] used CLE for prostate imaging ex vivo and in vivo. For our first two specimens, we followed
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Fig. 2 – Prostatic tissue. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
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Fig. 3 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology; RP = radical prostatectomy.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Fig. 4 – Prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
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Fig. 5 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Fig. 6 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
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Fig. 7 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
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Fig. 8 – Prostatic tissues. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
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Fig. 9 – Prostatic tissues. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
their protocol [12], but the saturation was too intense and
tissues were difficult to identify: excessive luminosity due
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Fig. 10 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron.
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Fig. 11 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Fig. 12 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
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Fig. 13 – Different aspects of prostate cancer. GS = Gleason score; HES = haematoxylin/eosin/saffron; RP = radical prostatectomy.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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to fluorescein made the acquired image overbrilliant. It was therefore impossible to identify intra- or extraprostatic tissue based on the images alone. When redipping into water, the images became interpretable. We therefore developed a modified protocol, as described in our “Patients and methods” section. Different timings between surgery and imaging can explain the different emissions of fluorescein staining; therefore, in order to avoid oversaturation, the optimised protocol is a 15 s–1 min protocol. When images were insufficient and/or undersaturated, a 1– 2 min protocol was adapted; however, as shown in our result section, this was likely to be more related to prolonged time between surgery and the CLE examination. One of the major difficulties at the beginning of our experience was the high magnification that the CLEGastroflex probe allows. Usually, prostate slides are examined under a microscope at a low magnification, in order to have a good overview of the tumour extension and architecture. With CLE examination, magnification is comparable with 800 at optic microscopy; therefore, analysis of a part of a gland or just one gland, when of small size, was possible. For this reason, only Gleason grades could be established. Therefore, especially for the first two specimens, having seen the tumour on a fresh frozen sample helped identify (tumour) glands at this high magnification. On the contrary, the pathologist had no difficulty in recognising all the tissues surrounding the prostate, and distinguishing the outer part from the inner part of the prostate was no major issue. This experience allowed us to differentiate between intra- and extraprostatic tissues, since several structures were easily recognisable, such as adipocytes, and fibrotic and connective tissues. It was possible to recognise vessels and nerves, but these findings need to be considered with caution, as our experience was performed ex vivo without blood flow. Based on the pathologist’s experience and expertise, among Gleason grade 4 glands, glomeruloid and cribriform glands were the easiest to recognise (Fig. 4). Based on morphology only, differentiating between Gleason grade 3 and normal prostatic glands is challenging in CLE imaging: small, well-defined, individual glands with different sizes can be mistaken for PCa or normal tissue [14]. However, most importantly, visualisation by CLE allowed us to distinguish between benign and malignant glands due to fluorescence: benign glands did not capture fluorescein in the cytoplasm of their cells, while tumour cells did (Fig. 4). Glands of Gleason grade 5 can be poorly differentiated or display solid aspects with comedonecrosis [14,15]. In case of poor differentiation, this pattern is difficult to distinguish with CLE: single cells enriched with fluorescein were observed in our study (Fig. 1). Overall, we believe that the significant value of this preliminary study allows pathologists and urologists to make a clear distinction between prostatic and periprostatic tissues. The sample size of our study is not a limitation, since we selected patients with the objective to collect representative images.
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In our study, the initial histological interpretation was made by a senior GU pathologist, and all CLE pictures were confirmed with HES staining of sampled tissues. It appears that the learning curve is short, especially regarding extraprostatic tissue interpretation. The in vivo experience will require a strong collaboration between pathologists and surgeons, with the pathologist being available during surgery on site or in remote connection with the operating room. However, we anticipate that urologists could be trained in CLE image interpretation and may, in future, be able to differentiate periprostatic from intraprostatic tissues. We can also foresee the creation of a large image database leading to the use of artificial intelligence software for image interpretation, making the process completely independent. Our study is unique due to the extensive description of CLE images of all prostatic and periprostatic tissues, providing a series of pictures that can be used as a model. The Martini Klinik group demonstrated that intraoperative frozen sections lead to an increased rate of nerve-sparing surgery, while decreasing the PSM rate, although the BCR rate remains stable [8]. The major limitation is the ex vivo nature of this study. Intraoperative distinction between tissues may become challenging in a highly magnified field on a mobile organ. Furthermore, the fluorescein administration protocol is not standardised yet. In order to avoid interindividual bias, extensive experience gained by a group of expert pathologists should be considered. 5.
Conclusions
We provide CLE images of peri- and intraprostatic tissues with different Gleason grades of PCa. Each CLE image was correlated with the corresponding HES final pathology image, allowing the edition of an atlas that will become a useful tool for GU pathologists and urologists interested in assessing the diagnostic performance of CLE during RP. When further in vivo studies will confirm its accuracy, CLE could become a valid intraoperative process for achieving negative SMs.
Author contributions: Bertrand Guillonneau had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Guillonneau, Compérat, Panarello. Acquisition of data: Compérat, Panarello. Analysis and interpretation of data: Compérat, Panarello. Drafting of the manuscript: Panarello, Compérat, Guillonneau. Critical revision of the manuscript for important intellectual content: Guillonneau, Compérat. Statistical analysis: Panarello. Obtaining funding: None. Administrative, technical, or material support: Seyde, Colau, Terrone. Supervision: None. Other: None.Financial disclosures: Bertrand Guillonneau certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding,
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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consultancies, honoraria, stock ownership or options, expert testimony,
[8] Schlomm T, Tennstedt P, Huxhold C, et al. Neurovascular structure-
royalties, or patents filed, received, or pending), are the following:
adjacent frozen-section examination (NeuroSAFE) increases nerve-
None.Funding/Support and role of the sponsor: None.
sparing frequency and reduces positive surgical margins in open and robot-assisted laparoscopic radical prostatectomy: experience after 11 069 consecutive patients. Eur Urol 2012;62:333–40.
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available at www.sciencedirect.com journal homepage: www.europeanurology.com/eufocus
Prostate Cancer
Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy Daniele Panarello a,b, Eva Compe´rat c, Olivia Seyde c, Alexandre Colau a, Carlo Terrone b, Bertrand Guillonneau a,* a
Service of Urology, Diaconesses—Croix St Simon Hospital, Sorbonne University, Paris, France;
b
Department of Urology, San Martino Hospital, Genoa
University, Genova, Italy; c Service of Pathology, Tenon Hospital, HUEP, Sorbonne University, Paris, France
Article info
Abstract
Article history: Accepted January 8, 2019
Background: Confocal laser endomicroscopy (CLE) is an optical device that aims to image histological architecture and may be used to reduce positive surgical margins. The ability of CLE to describe prostatic and periprostatic tissues, and prostate cancer (PCa) is still an object of investigation. Objective: To create an atlas of ex vivo CLE images of prostatic and periprostatic tissues, and PCa in order to recognise different prostatic structures. Design, setting, and participants: From November 2017 to February 2018, 15 patients underwent radical prostatectomy for biopsy-proven PCa. Outcome measurements and statistical analysis: Based on preoperative data and macroscopic examination, tumour location was assessed and confirmed on frozen sections. Prior to ex vivo CLE analysis, prostates were stained with fluorescein 10%. We used a GastroFlex probe to collect images of periprostatic tissue (adipose tissue, fibrous and connective tissues, vessels, nerve sheets, seminal vesicles, and urethra). Normal prostatic glands and tumour tissue according to the Gleason grade were analysed. Each PCa Gleason score was represented. Results and limitations: A total of 139 video clips and 237 pictures of prostatic and periprostatic tissues were collected. Among them, we selected 16 highly representative images. Adipose tissue, fibrous tissue, and connective tissue were supposable in all 15 specimens. PCa glands captured fluorescein in their cytoplasm, normal prostatic glands did not capture fluorescein, and glandular structures were easily recognisable. The principal limitation of this study is its ex vivo nature of the study. Conclusions: Each CLE image was correlated with the corresponding haematoxylin/ eosin/saffron definitive pathology image, allowing building of an atlas as a necessary tool to assess the diagnostic performance of CLE during radical prostatectomy in achieving negative surgical margins. Patient summary: In this study, we aim to provide an atlas of images illustrating prostatic, periprostatic, and PCa tissues obtained using Cellvizio confocal laser endomicroscopy as a tool for further interpretation of intraoperative surgical margins during radical prostatectomy. © 2019 Published by Elsevier B.V. on behalf of European Association of Urology.
Associate Gratzke
Editor:
Christian
Keywords: Confocal Laser Endomicroscopy Prostate Prostate Cancer
* Corresponding author. Service of Urology, Diaconnesses—Croix St Simon Hospital, 125 rue d’Avron, 75020, Paris, France. Tel.: +33 1 44 74 10 56. E-mail address: [email protected] (B. Guillonneau).
https://doi.org/10.1016/j.euf.2019.01.004 2405-4569/© 2019 Published by Elsevier B.V. on behalf of European Association of Urology.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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1.
Introduction
laparoscopic or robotic-assisted surgery was performed. No fluorescein was intravenously injected during surgery. All patients provided written
Prostate cancer (PCa) is the second most commonly diagnosed cancer in men. The incidence of PCa diagnosis in Western and Northern Europe is 94.9 and 85 per 100,000 age-standardised rate [1]. Different treatments are possible for PCa: for localised disease, the options are active surveillance, radical prostatectomy (RP), and radiation therapy. RP is an alternative to active surveillance for low-risk PCa patients who accept a trade-off between toxicity and prevention of disease progression; it is recommended in intermediate-risk patients and can be considered in high-risk PCa [1]. The goal of RP is complete removal of disease while preserving continence and potency whenever possible [2]. Following Walsh and Donker’s [3] initial study, preservation of neurovascular-bundles (NVBs) is mandatory to preserve potency and improve continence after RP. Despite the use of magnification in laparoscopic and robot-assisted RP, positive surgical margins (PSMs) continue to affect the oncological results. A recent literature review on the results of surgical techniques published between 2008 and 2011 reported a PSM prevalence ranging from 6.5% to 32%, with a mean value of 15% when not stratified by pathological stage. The overall mean PSM rate was 9% (range: 4–23%) in pT2, 37% (range: 29–50%) in pT3, and 50% (range: 40–75%) in pT4 cancers [4]. PSM status is correlated with biochemical recurrence (BCR) [5] and, even if still debated [6], could affect PCa-specific mortality [7]. In order to improve the rate of nerve-sparing surgery while reducing PSM rate, the Martini Klinik group published in 2012 their case series assessing the impact of the NeuroSAFE technique on PSM, BCR, and nerve sparing [8]. NeuroSAFE uses intraoperative frozen sections to determine whether a nerve-sparing approach is possible. Nowadays, many technologies aim to increase the visual capability and define cellular architecture in vivo. The confocal laser endomicroscopy (CLE) system Cellvizio (Manua Kea Technologies, Paris, France) is an optical device based on a 488-nm blue laser used in combination with fluorescein, providing high-resolution imaging of spatial cellular disposition similar to histopathology [9]. Presently, it is used for bladder, upper urinary tract [9], gastrointestinal tract [10], and lung [11] tumours. CLE was preliminary tested during robot-assisted RP, and tissues were sampled ex vivo and in vivo without extensive description of different tissues analysed [12]. The aim of our study was to create an atlas of ex vivo prostate tissue images using CLE, analysing periprostatic tissue, normal prostatic tissue, and PCa images to provide an illustrated guide useful for future intraoperative evaluation of surgical margin (SM) status during RP. The rational was to recognise tumour tissue in case of tumour-positive margins, and to differentiate from nontumoural intraprostatic margins. 2.
Patients and methods
From November 2017 to February 2018, we selected 15 patients eligible for RP after biopsy-proven PCa with different preoperative Gleason
informed consent. All prostate specimens were immediately preserved fresh within a tissue-safe vacuum-sealed package in the operative room, which was delivered to the pathology laboratory, where packages were unsealed when the study examination started. A senior genitourinary (GU) pathologist assessed the tumour location based on macroscopic examination, preoperative prostate magnetic resonance imaging (MRI) results, and positive biopsy topography, and then performed targeted frozen sections. The fresh frozen procedure was performed according to the pathology standards in order to confirm the presence of PCa, and the slices were stained by haematoxylin/eosin/saffron (HES). In order to recognise the examined areas on definitive histopathological samples, different coloured inks were used to stain the areas of interest. The staining protocol is shown in Table 1. We first focused on periprostatic tissue to identify the outer layer of the prostate (adipose, fibrous, and connective tissue); we then evaluated the structures of the outer layer such as vessels, nerve sheets, seminal vesicles, and urethra. Furthermore, we aimed to recognise normal prostatic glands and tumour glands. The Gleason score was used according to the World Health Organization (WHO) 2016 classification. We utilised a GastroFlex probe for imaging. The technical specifications are as follows: maximal field of view 240 mm, depth 55–65 mm,
lateral resolution 1 mm, and magnification comparable with 800 at optic microscopy. Regarding fluorescein-staining protocol, for the first two cases, we used the protocol previously published [12], which consisted of 2-min dipping in a solution of fluorescein and 7-min washing in saline solution. This was then modified for a protocol of 10 ml fluorescein 10% (100 mg/ ml) and 500 ml of saline solution. We dipped the prostate in this solution for 15 s followed by 60 s in water. Prostates examined after >12 h from surgery had poorer image quality and the fluorescein dipping was insufficient; the specimens were therefore immersed in the fluorescein solution for 60 s followed by 120 s of washout. Every sequence was video recorded, as well as the time of relevant images together with their spatial distribution. All videos were reviewed by the pathologist and the urologist. The images and findings were correlated with the final pathology. The correlation was made with the use of different coloured inks, which marked the areas where the videos were recorded. After the classical histopathology handling, we were able to find the exact location of the initial video, and superposition
was made easily.
Table 1 – Staining protocol.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time from surgery
Stain protocol
Number of pictures
Number of clips
12:00 14:00 12:00 1:00 12:00 12:00 1:00 1:00 14:00 12:00 1:00 14:00 12:00 16:00 1:00
2 + 7 min 2 + 7 min 60 + 120 s 15 + 60 s 60 + 120 s 60 + 120 s 15 + 60 s 15 + 60 s 60 + 120 s 60 + 120 s 15 + 60 s 60 + 120 s 60 + 120 s 60 + 120 s 15 + 60 s
7 10 18 14 34 16 17 17 8 9 11 16 14 26 20
2 3 5 13 14 6 6 9 6 7 13 8 12 17 18
scores. We analysed the prostate specimens using CLE ex vivo. Either
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3.
Results
The final Gleason scores of PCa after standard histopathological techniques, used according to WHO 2016 recommendations [13], were as follows: one specimen with Gleason score 6 (3 + 3), five with Gleason score 7 (3 + 4), three with Gleason score 7 (4 + 3), four with Gleason score 8 (4 + 4), and one with Gleason score 9 (5 + 4) prostate adenocarcinoma. One patient, who
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presented a Gleason score of 9 (5 + 4) on the biopsy, was not graded due to neoadjuvant hormonal treatment leading to morphological alterations; nevertheless, the images were examined and recorded (Fig. 1). Patient characteristics are shown in Table 2. The delay between surgery and CLE imaging examination ranged from 1 to 18 h. Five prostate specimens were analysed within 1 h from surgery, and 10 specimens were
Fig. 1 – Periprostatic tissue. HES = haematoxylin/eosin/saffron.
Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004
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Table 2 – Patient characteristics.
PSA prebiopsy Clinical stage
Biopsy Gleason score
Biopsy ISUP grade group
Gland volume Pathological stage
Pathological Gleason score
Pathological ISUP grade group
Max tumour diameter Number of video clips per specimen Number of pictures per specimen
Mean
Median
9.75
7.36
cT1c cT2a cT2b cT2c cT3a 6 7 9 1 2 3 5 42.9
%
3 6 5 0 1 3 10 2 3 8 2 2
20.0 40.0 33.3 0.0 6.7 20.0 66.7 13.3 20.0 53.3 13.3 13.3
6 4 5 1 8 4 1 1 5 3 4 1
7.1 57.1 28.6 7.1 7.1 35.7 21.4 28.6 7.1
42.5
pT2 pT3a pT3b 6 7 8 9 1 2 3 4 5 23 9 16
n
20 8 16
ISUP = International Society of Urological Pathology; PSA = prostatespecific antigen.
analysed at >12 h (range 1—18 h). All frozen section slices were positive for PCa. We collected 139 different video clips (mean: nine clips per specimen) and 237 pictures (median: 16 pictures per specimen) of prostatic and periprostatic tissues. The mean time of video recording per patient was 8.53 min (range 2–29 min). Among the recorded sequences, we identified 237 pictures relevant for the CLE PCa atlas. Among them, we selected 52 informative images illustrating the different aspects of prostate tissue and PCa (Figs. 1–13). Among them, we superselected 16 very informative images of periprostatic tissues, prostatic tissues, and PCa (Figs. 1–4). The remaining 36 images (Figs. 5–13) were included in the atlas. At first, the outer layers of all 15 prostate specimens were examined. Adipose tissue, fibrous tissue, and connective tissue were comparable in all 15 specimens. Vessels and nerves were more difficult to distinguish from each other, but structures corresponding to nerve sheets and vessels were found and reported. Then, we examined the prostate apex and the surrounding muscular and urethral tissues, which displayed an excellent visual correlation with HES staining. Both cellularity and spatial distribution were visually comparable with the final pathology (Figs. 2 and 5 –7). Once the pathologist was trained in working with high magnification, glandular nontumoural intraprostatic tissues became easily recognisable (Figs. 3, 8, and 9). Periprostatic tissues were recognised and distinguished in CLE images since their histological architecture was perfectly comparable with the architecture of HES staining.
Adipocytes appear like large, black, round, nonfluorescent uniform cells with clear membranes (Figs. 2, 5, and 6). Smooth muscle is composed of multiple “cigar”-shaped cells (Figs. 2 and 5 –7); connective tissue surrounding the prostate is recognisable by the shape of the fibres within the extracellular matrix (Figs. 2 and 5 –7). Prostatic apex is composed of muscular cells, connective tissue, and rare glands. Nerves were recognisable especially in longitudinal sections. Regarding seminal vesicles, irregularity of the glandular lumen together with the presence of smooth muscle cells guided the interpretation of images. Basal cell hyperplasia was also observed (Fig. 3). These cells never displayed any fluorescence; nevertheless, they allowed us to differentiate between intra- and extraprostatic tissues. We were able to distinguish normal prostate glands from tumour tissue by fluorescence: PCa glands were identified for their early ability in capturing fluorescein in their cytoplasm, while normal prostatic glands did not capture fluorescein (Figs. 1, 3, 4, 8 –13). Regarding the various Gleason grades, we found either well-defined glands with cytoplasmic fluorescein emission corresponding to well-differentiated PCa of Gleason grade 3 or less well-differentiated aspects. The most easily recognisable Gleason grade was Gleason grade 4, especially glomeruloid and confluent glands with rigid patterns. Intraglandular tumour cells were easily seen, and some had papillae-like propulsions. The patient treated with neoadjuvant hormonotherapy also displayed easily recognisable aspects: the glands were of bigger size with a slightly fluorescent content, with the surrounding tumour glands capturing fluorescein as well. The most difficult correlation was related to poorly differentiated tumour glands; nevertheless, the latter showed some emission of fluorescein as well. 4.
Discussion
Images of prostatic and periprostatic tissues provide good CLE images to be used by pathologists and surgeons when starting their experience with CLE during RP. Presently, the decision to perform nerve-sparing surgery, either complete or partial, uni- or bilaterally, or to perform full resection of the neurovascular bundle is mainly based upon preoperative clinical features: prostate-specific antigen test, clinical stage, biopsy results, and prostate MRI analysis [1]. We do not yet have tools to assess the SM status intraoperatively; according to the European Association of Urology guidelines, intraoperative frozen section can be helpful in making the decision of whether to perform a nerve-sparing surgery [1]; nevertheless, this can lead to a less accurate definitive pathology analysis. For this reason, there is a rationale for tools on surgeon guidance during NVB dissection, ensuring first of all negative SMs and the optimal nerve-sparing approach while optimising oncological safety. We identified an improved protocol for ex vivo imaging based on a staining process in a fluorescein 0.2% solution. To date, only Lopez et al [12] used CLE for prostate imaging ex vivo and in vivo. For our first two specimens, we followed
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Fig. 2 – Prostatic tissue. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
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Fig. 3 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology; RP = radical prostatectomy.
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Fig. 4 – Prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
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Fig. 5 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
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Fig. 6 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
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Fig. 7 – Periprostatic tissues. HES = haematoxylin/eosin/saffron.
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Fig. 8 – Prostatic tissues. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
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Fig. 9 – Prostatic tissues. CLE = confocal laser endomicroscopy; HES = haematoxylin/eosin/saffron.
their protocol [12], but the saturation was too intense and
tissues were difficult to identify: excessive luminosity due
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Fig. 10 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron.
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Fig. 11 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
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Fig. 12 – Different aspects of prostate cancer. HES = haematoxylin/eosin/saffron; ISUP = International Society of Urological Pathology.
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Fig. 13 – Different aspects of prostate cancer. GS = Gleason score; HES = haematoxylin/eosin/saffron; RP = radical prostatectomy.
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to fluorescein made the acquired image overbrilliant. It was therefore impossible to identify intra- or extraprostatic tissue based on the images alone. When redipping into water, the images became interpretable. We therefore developed a modified protocol, as described in our “Patients and methods” section. Different timings between surgery and imaging can explain the different emissions of fluorescein staining; therefore, in order to avoid oversaturation, the optimised protocol is a 15 s–1 min protocol. When images were insufficient and/or undersaturated, a 1– 2 min protocol was adapted; however, as shown in our result section, this was likely to be more related to prolonged time between surgery and the CLE examination. One of the major difficulties at the beginning of our experience was the high magnification that the CLEGastroflex probe allows. Usually, prostate slides are examined under a microscope at a low magnification, in order to have a good overview of the tumour extension and architecture. With CLE examination, magnification is comparable with 800 at optic microscopy; therefore, analysis of a part of a gland or just one gland, when of small size, was possible. For this reason, only Gleason grades could be established. Therefore, especially for the first two specimens, having seen the tumour on a fresh frozen sample helped identify (tumour) glands at this high magnification. On the contrary, the pathologist had no difficulty in recognising all the tissues surrounding the prostate, and distinguishing the outer part from the inner part of the prostate was no major issue. This experience allowed us to differentiate between intra- and extraprostatic tissues, since several structures were easily recognisable, such as adipocytes, and fibrotic and connective tissues. It was possible to recognise vessels and nerves, but these findings need to be considered with caution, as our experience was performed ex vivo without blood flow. Based on the pathologist’s experience and expertise, among Gleason grade 4 glands, glomeruloid and cribriform glands were the easiest to recognise (Fig. 4). Based on morphology only, differentiating between Gleason grade 3 and normal prostatic glands is challenging in CLE imaging: small, well-defined, individual glands with different sizes can be mistaken for PCa or normal tissue [14]. However, most importantly, visualisation by CLE allowed us to distinguish between benign and malignant glands due to fluorescence: benign glands did not capture fluorescein in the cytoplasm of their cells, while tumour cells did (Fig. 4). Glands of Gleason grade 5 can be poorly differentiated or display solid aspects with comedonecrosis [14,15]. In case of poor differentiation, this pattern is difficult to distinguish with CLE: single cells enriched with fluorescein were observed in our study (Fig. 1). Overall, we believe that the significant value of this preliminary study allows pathologists and urologists to make a clear distinction between prostatic and periprostatic tissues. The sample size of our study is not a limitation, since we selected patients with the objective to collect representative images.
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In our study, the initial histological interpretation was made by a senior GU pathologist, and all CLE pictures were confirmed with HES staining of sampled tissues. It appears that the learning curve is short, especially regarding extraprostatic tissue interpretation. The in vivo experience will require a strong collaboration between pathologists and surgeons, with the pathologist being available during surgery on site or in remote connection with the operating room. However, we anticipate that urologists could be trained in CLE image interpretation and may, in future, be able to differentiate periprostatic from intraprostatic tissues. We can also foresee the creation of a large image database leading to the use of artificial intelligence software for image interpretation, making the process completely independent. Our study is unique due to the extensive description of CLE images of all prostatic and periprostatic tissues, providing a series of pictures that can be used as a model. The Martini Klinik group demonstrated that intraoperative frozen sections lead to an increased rate of nerve-sparing surgery, while decreasing the PSM rate, although the BCR rate remains stable [8]. The major limitation is the ex vivo nature of this study. Intraoperative distinction between tissues may become challenging in a highly magnified field on a mobile organ. Furthermore, the fluorescein administration protocol is not standardised yet. In order to avoid interindividual bias, extensive experience gained by a group of expert pathologists should be considered. 5.
Conclusions
We provide CLE images of peri- and intraprostatic tissues with different Gleason grades of PCa. Each CLE image was correlated with the corresponding HES final pathology image, allowing the edition of an atlas that will become a useful tool for GU pathologists and urologists interested in assessing the diagnostic performance of CLE during RP. When further in vivo studies will confirm its accuracy, CLE could become a valid intraoperative process for achieving negative SMs.
Author contributions: Bertrand Guillonneau had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Guillonneau, Compérat, Panarello. Acquisition of data: Compérat, Panarello. Analysis and interpretation of data: Compérat, Panarello. Drafting of the manuscript: Panarello, Compérat, Guillonneau. Critical revision of the manuscript for important intellectual content: Guillonneau, Compérat. Statistical analysis: Panarello. Obtaining funding: None. Administrative, technical, or material support: Seyde, Colau, Terrone. Supervision: None. Other: None.Financial disclosures: Bertrand Guillonneau certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding,
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consultancies, honoraria, stock ownership or options, expert testimony,
[8] Schlomm T, Tennstedt P, Huxhold C, et al. Neurovascular structure-
royalties, or patents filed, received, or pending), are the following:
adjacent frozen-section examination (NeuroSAFE) increases nerve-
None.Funding/Support and role of the sponsor: None.
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Please cite this article in press as: Panarello D, et al. Atlas of Ex Vivo Prostate Tissue and Cancer Images Using Confocal Laser Endomicroscopy: A Project for Intraoperative Positive Surgical Margin Detection During Radical Prostatectomy. Eur Urol Focus (2019), https://doi.org/10.1016/j.euf.2019.01.004