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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
230 M. M. Shevchuk
(a) (b) (c)
(d) (e) (f)
Figure 9. Images of non-papillary kidney tumors: MPM image (a) and corresponding H&E image (d)
of low-grade clear-cell RCC with sheets of cells (color-coded green), separated by delicate branching
network of vascular tissue (color-coded red). Inset shows tumor cells with fat droplets (arrow: colorcoded green)in the cytoplasm surrounding nucleus. MPM image (b) and corresponding H&E image (e)
of high-grade clear-cell RCC with tumor cells (color-coded green) and stroma (color-coded red). Inset
shows cells with homogeneous cytoplasm (color-coded green) lacking fat droplets. MPM image (c)
and corresponding H&E image (f) of chromophobe RCC with sheets of tumor cells (color-coded green)
and thickened blood vessels (arrows). Inset shows cells with prominent intra-cytoplasmic granules
(color-coded blue), hypothesized to correspond to cytoplasmic vesicles. Adapted from Ref. [24].
et al.
tissues, without using other ancillary studies.27The clinical benefit of this
is that the distinction between a benign and a malignant oncocytic tumor
can be made more quickly and less expensivelythan is done currently, thus
avoiding the need to perform multiple immunohistochemical and ancillary
studies.
Spectroscopic studies using Raman spectroscopy, optical reflectance
spectroscopy, and optical emissions spectroscopy have been performed
ex vivo on renal neoplasms and found to be successful in differentiating tumor from normal kidney parenchyma and in distinguishing benign
Genitourinary System 231
from malignant tumors.
28,29
These technologies have not been translated
to in vivo evaluation.
Another potential application of IVM is in renal transplantation
medicine. A study by Andrews et al. describes the use of OCT and Dopplerbased OCT (DOCT) to image donor kidneys ex vivo prior to transplantation
and in vivo after transplantation. The kidneys which showed a diminished
proximal tubular diameter reflected acute ischemic damage and risk of
post-transplantation acute tubular necrosis.
30
Prostate
Prostate cancer is the second cause of death among men in the United
States and comprised 26% of cancer diagnoses in men in 2015.
diagnosis and determination of appropriate management strategies remain
31
challenging givendisease heterogeneity.
The clinical dilemma in prostate
cancer management arises from the difficulty in distinguishing aggressive
prostatic cancers from indolent tumors, which account for a significant
portion of cancers identified as a result of prostate-specific antigen (PSA)
screening. Recent guidelines recommendthat low-risk (Gleason 3+3), lowvolume prostate cancers may be carefully followed via active surveillance
in appropriately selected patients.
31
In order to appropriately select patients
for active surveillance, diagnostic needle biopsies must not only identify
the presence of prostatic carcinoma but also identify the highest Gleason
grade present. Prostate needle biopsies are performed in a standard 12-core
template pattern with ultrasound guidance. These biopsies rarely have identifiable foci to target on ultrasound and may miss up to 30% of cancers.
Recent fusion imaging technologies (MRI image overlayof intraprocedural
ultrasound) show promise in identifying clinically significant tumor foci
while minimizing diagnosis of indolent disease.
31
Trials are underway to
further facilitate the identification of prostatic carcinoma at the time of
needle biopsy, either in vivo or ex vivo, to improve staging and minimize
morbidity.
OCT has been studied ex vivo in resected prostates in the form of an in-
tissue, needle-basedimaging platform to determine co-registration between
3
Accurate
32
232 M. M. Shevchuk
(a)
(b)
et al.
(c)
Figure 10. Ff-OCT of prostate core needle biopsies. A single image (a) is produced for each biopsy
core (b), and zooms can be performed on aggregated tumor glands Gleason 3 + 3 (c, d). Adapted
from Ref. [34].
(d)
needle-based OCT diagnosis and histopathology with demonstrated
adequate correlation.
33
In another study, ff-OCT showed an overall accuracy for ex vivo diagnosis of freshly biopsied, unprocessedneedle core biopsies of the prostate to be 82% and the ability to accurately grade tumors
of Gleason score >3 + 4 to be 72% of the time (Figure 10).
34
A feasibility study conducted using video-rate fluorescence structured illumination microscopy intraprocedurally on fresh prostate needle core biopsies,
immersed for a few seconds in acridine orange, reported a sensitivity of
63–88% and a specificity of 78–89% overall for prostate cancer detection. When there was more than 5% tumor within the core, however, the
sensitivity rose to 75–95%.
35
Another possible new application of IVM
Genitourinary System 233
technology was reported by Nguyen et al. They used an interferometry
system of quantitative phase imaging and machine learning to achieve 82%
accuracy for computer-generated diagnosis of Gleason grade 3 carcinoma
vs. Gleason grade 4 tumor.
36
IVM technologies may also contribute to improved surgical treatment of prostate cancer. Intraoperative histopathologic data may serve a
role in evaluating surgical margins during radical prostatectomy or localized therapy, as margin status guides post-prostatectomy care and has
37
been demonstrated to impact disease progression.
Intraoperative IVM
can also assist in identifying periprostatic nerves to facilitate nerve-sparing
prostatectomy.
CLE was employed by Lopez et al. during robotic radical prostate-
ctomy to identify the periprostatic neurovascular bundles and to ascertain
38
their viability after dissection of the prostate (Figure 11).
The advantage
of this IVM use was that the CLE instrument was compatible with the da
Vinci Surgical System (Intuitive Surgical, Inc) and that CLE helped identify
periprostatic nerves with the aim of preserving potency. The same group
used CLE in the ex vivo setting to identify focal extraprostatic extension,
demonstrating the feasibility of using CLE ex vivo.
Another IVM technology that shows promise for evaluating the entire
resection margin of a radical prostatectomy specimen, which can be done
in real time, is video-rate structured illumination microscopy (VR-SIM).
39
This technology successfully identified positive margins intraoperatively
in 3 of the 4 cases, which were confirmed to be positive by histopathology. In one additional case, VR-SIM identified a positive margin, which
was initially missed by histopathology, because of the limitations of sectioning. In this last case, the VR-SIM diagnosis contributed to the patient’s
post-operative care.
Light-sheet microscopy is another technology which can give highresolution 3D images of fresh, whole radical prostatectomy 4 mm-thick
gross sections (Figure 12).
40
This technology could be used immediately
afterprostate resection orlater in the specimengrossing process to imagethe
entire prostatein three dimensions for marginpositivity and to identifyareas
of the highest Gleason grade. Light-sheet microscopy has also been studied
40
to image needle core biopsies in real-time ex vivo.
Light-sheet microscopy
shows three-dimensional histology of prostatic carcinoma, which not only
234 M. M. Shevchuk
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 11. CLE images of the neurovascular bundle (NVB). Nerve axons visualized with 0.85 mm probe (a) and 2.6 mm probe (b–g). Nerves were
visualized before (b) and after (c, d) NVB dissection. Residual ner ve structures were present on prostatic capsule after neurovascular dissection (e).
Intact NVB seen ex v ivo on non-nerve-sparing prostate specimen (f). Panoramic image of NVB generated with mosaicing algorithm from images obtained
during in vivo CLE, with erythrocytes within blood vessels on left and nerve fibers on right (g). Demonstration of CLE probe use intraoperatively within the
robotic system and corresponding CLE image of the NVB (h). Adapted from Ref. [38].
et al.
Genitourinary System 235
(a) (b)
(c)
Figure 12. Light-sheet microscopy of prostate specimen after radical prostatectomy. (a) The tissues
are processed and then imaged at a speed of v = 50 s/cm
of h = 320µm to accommodate for any surface irregularities and tilting errors. A horizontal (en face)
2D “section” from the 3d dataset is shown on the top left, prior to surface extraction, where regions of
defocus and incomplete imaging are seen (inset arrows). On the bottom right, the irregular surface of
the large specimen has been digitally extractedfrom the 3D dataset to provide a comprehensive image
of the surface. (b) Moderate- and high-magnification images of normal prostate glands, where a layer
of both basal and epithelial cells is observed (inset arrows). A corresponding H&E histology image is
shown on the right. (c) A region with benign prostate (left) transitioning into prostate adenocarcinoma
(right), which exhibits a crowding of glands with a single epithelial cell layer (inset arrow). Adapted
from Ref. [40].
2
, which provides a vertical field of view
helps with exact Gleason grading intraprocedurally, but also has the potential for a new field of three-dimensional histopathology.
A feasibility study of MPM technology using freshly resected radical prostatectomy specimens showed that MPM accurately identifies periprostatic structures (nerves, blood vessels, and capsule), as well as benign
prostatic glands and prostatic carcinoma (Figure 13).
41
Gleason grade was
also identifiable in most tumor foci. This technology can similarly be used
to image core needle biopsies of the prostate.
Spectroscopic technologies are also being employed to identify carcinoma in radical prostatectomy specimens. They do not give
histopathology-like images but rather identify areas in the prostate which
contain carcinoma. A combined auto-fluorescence and light reflectance
236 M. M. Shevchuk
(a) (b) (c)
Figure 13. MPM imaging of the prostate and periprostatic tissue. (a) Higher magnification image of
a small nerve bundle at the surgical margin, showing fluorescence that derives from the axoplasm or
the cytoplasm of the Schwann cells. A similar view of the nerve using CLE can be seen in Figure 11,
(d). (b, c) Low-magnification images of human prostate gland showing prostate cancer. In the MPM
image (panel b), two regions can be identified: small glands can still be distinguished (a, Gleason
grade 3) and sheets of fused glands with no glandular architecture or intervening stroma (b, Gleason
grade 4). Panel c shows an H&E-stained image from a corresponding area. Adapted from Ref. [41].
et al.
spectroscopy feasibility study of freshly resected whole radical prostatectomy specimens successfully identified Gleason score 7, 8, and 9 carcinomas in 91.1%, 91.9%, and 94.3% of tumor foci within the prostates and
42
within the prostatic capsules.
Raman spectroscopy is also being investigated as a tool for identifying carcinoma in radical prostatectomy specimens
and in biopsies.
43
Testis
IVM technology is also applicable in fertility medicine. Feasibility studies
have been conducted in patients with non-obstructive azoospermia to identify seminiferous tubules containing viable sperm. The ability to identify
sperm in the testes of these patients may impact the success rate of assisted
reproduction.
CLE use in testis imaging was demonstrated in a feasibility study as a
means of identifyingviable spermatids in testicular tubules (Figure 14).
Resected testes from hormonally treated transsexual patients were imaged
with the use of fluorescent dyes. Rare intratubular spermatids were identified in 30% of patients, whereas spermatozoa were present in all. The
authors concluded that in the future CLE might be used in azoospermic
44,45
Genitourinary System 237
(a) (b)
Figure 14. Longitudinal view of human testicular tubules as seen by CLE before (a) and after (b) staining with acriflavine. Due to the treatment with estrogens and anti-androgens, mainly spermatogonia
are visible. Adapted from Ref. [44].
men to identify areas of testes which contain sperm in order to achieve
better results with in vitro fertilization.
An MPM ex vivo imaging study showed an 86% concordance between
the MPM identification of intratubular spermatids as compared with the
46
corresponding H&E-stained histologic sections.
MPM was also able to
determine that the tubular diameters in azoospermic patients were diminished, which was confirmed on H&E histology.
Future Perspectives
IVM technologies show promise for numerous clinical applications in urology. In the urinary tract, IVM can facilitate identification of foci of the
highest grade and stage of urothelial carcinoma for more definitive diagnosis and treatment. Renal tumors can be assessed using IVM to distinguish
benign and malignant tissues and to help secure negative surgical margins,
which could impact recurrence in high-risk patients.
tion could benefit from IVM imaging to better identify patients at risk of
ATN and reduced post-transplantation function. IVM may facilitate evaluation of high-grade tumor foci on prostate biopsy, as well as identification of
surgical margins and relevant anatomy during radical prostatectomy. IVM
may also play a role in fertility medicine to identify viable sperm for in vitro
fertilization.
47
Renal transplanta-
238 M. M. Shevchuk
(a) (b) (c)
Figure 15. MPM imaging of the testicle. Seminiferous tubular histology patterns imaged by MPM at
low (a) and high (b) magnification compared to high magnification stained tissue (c). Note normal
spermatogenesis (green areas) (a–c) (from Figure 14. Adapted from Ref. [46].
et al.
In parallel, these technologies may be broadly applied ex vivo in genitourinary pathology. It can be used to ascertainadequacy of diagnostic biopsies, be they from the prostate, the kidneys, the urinary tract, or metastatic
sites. This technology may allow pathologists to more definitively evaluate
large specimens, be they prostate, bladder, or kidney, for margin involvement. Additional exciting new perspectives for IVM technologies involve
their use to acquire three-dimensional histopathology and to visualize blood
flow patterns, which may result in additional diagnostic information and
criteria. Although IVM imaging of the genitourinary tract remains in its
infancy, multiple feasibility studies are being currently conducted and they
will likely continue to grow in use for specific indications in urology and in
genitourinary pathology, using the specific best technology for each indication. In vivo and ex vivo microscopy will become incorporated into the
rapidly expanding fields of digital imaging/digital pathology, with appropriate uses of AI, resulting in more rapid and more definitive diagnoses,
and better outcomes for patients with genitourinary diseases.
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