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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
310 D. R. Fels Elliott & A. M. Amacher
imaging parameters can be optimized using different ex vivo microscopy
technologies in relevant clinical settings.
Intraoperative Evaluation of Surgical Margins
Intraoperative evaluation of surgical margins for the presence of tumor
is most often performed using frozen tissue sections with histological
stains such as hematoxylin and eosin (H&E) or toluidine blue. Limitations of frozen section analysis include damage or loss of tissue from the
specimen, additional time taken to freeze and stain tissue, freezing artifact including distortion of architecture, and risk of less accurate diagnosis in comparison to formalin-fixed paraffin-embedded (FFPE) tissue.
Ex vivo microscopy has been proposed as a rapid alternative to frozen
section analysis using morphological and functional imaging technologies. Morphological imaging techniques involve the characterization of
cellular and subcellular structures within tissue using parameters such as
shape and density.
(CLSM), optical coherence tomography (OCT), photoacoustic imaging,
two-photon excited fluorescence microscopy, a nd second-harmonic generation microscopy. In contrast, functional imaging technologies, such
as fluorescence lifetime imaging microscopy (FLIM) and Raman-based
microscopy, visualize physiologic parameters such as differences in blood
flow, metabolism, or chemical composition. Ex vivo microscopy has gained
particular interest from clinicians regarding the intraoperative evaluation of
margins for breast and skin specimens, and these sites will be the focus of
the following sections.
1
Examples include confocal laser scanning microscopy
Applications in breast conservation surgery
Ductal carcinoma in situ (DCIS) and early-stage breast cancers are often
treated with breast conservation surgery (BCS) and radiation. The rate of
positivemargins in BCS specimens variesfrom 4% to 20%
with larger tumor size, multifocality, and the presence of microcalcifica-
6
tions.
Re-excision is generally advised for patients with positive margins
because the risk of local tumor recurrence is two to three-fold higher.
2–5
and correlates
7,8
Ex Vivo
Applications 311
Revision surgery may also be recommended for “close” negative margins
that are less than 1 mm or 1–2 mm,
9
so it is desirable for the surgeon
to know intraoperatively whether the tumor is at least 2 mm away from
the specimen margin. Overall, re-excision rates for patients undergoing
BCS range from 11–46%.
10
To address this issue and improve patient care,
intraoperative adjuncts such as frozen section analysis, touch preparation
(imprint) cytology, and specimen imaging technologies (macroscopic and
microscopic) have been proposed. As discussed above, the limitations of
frozen section analysis include loss of tissue and longer operation times.
An additional challenge is an increased false negative rate in BCS patients
8
who receive neoadjuvant chemotherapy.
Cytologic evaluation with touch
preparations has limited utility in BCS specimens because it only samples
surface cells and cannot reliably distinguish between in situ and invasive
carcinoma. Macroscopic specimen radiography is performed routinely at
many institutions and is a useful adjunct to visualize a tumor mass lesion,
biopsy clip, or microcalcifications, but generallylacks the resolutionneeded
to detect microscopic tumor foci. Several ex vivo microscopic imaging
technologies have been proposed to evaluate breast specimen margins,
including spectroscopic methods (e.g. radiofrequency spectroscopy, diffuse
reflectance spectroscopy, intrinsic fluorescence spectroscopy, and Raman
spectroscopy),interferometric methods (e.g. optical coherencetomography
and interferometric synthetic aperturemicroscopy) and others. Collectively,
these technologies aim to maximize tissue penetration to a depth of at least
1–2 mm while maintaining high image resolution. Other high-resolution
imaging modalities with potential utility for intraoperative breast margin
assessment include energy-dispersive X-ray diffraction computed tomog-
11
raphy (EDXRDCT),
tomography (Micro-CT).
high-frequency ultrasound,12and micro-computed
13
Radiofrequency spectroscopy — The MarginProbe
The MarginProbe(Dune Medical Devices, Boston, MA, USA) is the
first FDA-approved, commercially available device for margin assessment
in breast conservation surgery.
14
The device consists of a detachable,
312 D. R. Fels Elliott & A. M. Amacher
Figure 1. The MarginProbeis commerciallyavailablefor use i n breast conservation surgery and uses
radiofrequency spectroscopy to evaluate electrical properties of breast tissue. The device issues a
binary positive or negative result for each measurement taken within a 7 mm area. Image reprinted
with permission.
15
hand-held,∼1.6 cm-diameter probe anda control console that uses radiofrequency spectroscopy to rapidly assess the electrical properties of breast tissue (Figure 1).
10,15
The device generates radiofrequency electromagnetic
fields that are differentially absorbed and reflected by tumor and normal
breast tissue due to alterations in nuclear size, increased vascularity, and
stromal density. Each measurement is made over a 7 mm-diameter area to a
depth of 2–3 mm within one to five seconds. Based on predetermined tissue
signatures for tumor and normal breast tissue, the MarginProbe
gives a
“positive” or “negative” result for each scan with a sensitivity of 70% and
specificity of 70% (MAST study, N = 76 patients).
16
The sensitivity of
detection was limited by the ability of the device to detect very small tumor
foci (sensitivity 56% for 0.7 mm foci) in comparison to larger tumor nests
(sensitivity 97% for 6.6 mm foci). The efficacy of the MarginProbe
has
been evaluated in prospective, randomized, controlled clinical trials, including the US Pivotal Study (N = 300 patients)
17
and the German Multicenter
Ex Vivo
Figure 2. An optical spectral imaging probe developed by Wilke et al. uses diffuse reflectance
spectroscopy to measure optical scatter of beta carotene in breast tissue. A negative margin, positive
margin for ductal carcinoma in situ (DCIS), and positive margin for invasive carcinoma are depicted.
Blue areas correlate with benign breast tissue, while red areas correspond to in situ or invasive
carcinoma. Image reprinted with permission.
Applications 313
19
Study (N = 596 patients),18which showed the device was a useful adjunct
to guide additional excision of positive margins in BCS specimens. In both
trials, the MarginProbe
device improved the intraoperative detection of
positivemargins and decreased the number of patients requiring re-excision
procedures. The US Pivotal Study showed re-excision rates of 5.6% in the
device arm versus 12.7% in the control arm, a nd the German Multicenter
Study showed 19.8% in the device arm versus 25.8% in the control arm.
Optical spectroscopy
Diffuse reflectance spectroscopy(DRS) takes advantage of intrinsic optical
contrast sources within breast tissue, including morphologic (cell density, beta-carotene within adipocytes, a nd collagen content) and physiologic (oxygenated and deoxygenated hemoglobin content) properties to
detect spectral signatures from absorbance and scattering patterns of visible and near-infrared light.
oped a hand-held optical spectral imaging probe with a xenon light source
(450–600 nm) capable of imaging a 3 × 1 cm area of tissue to a depth of
2.2 mm within ∼25 s (Figure 2).
of in situ and invasive breast malignancies, the probe accurately identified
close or positive margins in 79.4% of lumpectomy specimens.
19,20
Using this technology, Wilke et al. devel-
19
In a cohort of 48 patients with a range
19
Imaging a
314 D. R. Fels Elliott & A. M. Amacher
larger cohort of 70 patients yielded a sensitivity of 74% and specificity of
21
86%.
Increased collagen content correlated with increased variability in
the scattering of light, and the ability of the optical spectral imaging device
to differentiate benign from malignant tissue was markedly improved in
postmenopausal women.
21
Lue et al. also described a multimodal optical
spectral imaging probe that combines DRS with intrinsicfluorescence spectroscopy (IFS) to obtain high-resolution images spanning a larger field of
view (10 × 10cm) within 20 min.
22
Preliminary results using human breast
tissue showed that small tumor foci (<1–3mm) were detected by the combined DRS and IFS probe, while individual DRS and IFS maps separately
failed to identify these tiny foci. Broadband reflectance spectroscopy is
another optical platform to detect scattering signatures in breast tissue.
23,24
Laughney et al. showed that localized scattering signatures distinguished
between benign and malignant breast tissues with 93% sensitivity and 95%
specificity in a cohort of 32 patients.
24
Raman spectroscopy
Raman spectroscopy measures molecular vibrations of tissue components
such as fat, collagen, beta-carotene, calcium, cellular cytoplasm, and nuclei
with a sampling volume of approximately 1 mm
spectra show differences in chemical and morphologic composition of
benign and cancerous breast tissue (Figure 3).
fat and collagen were significant parameters in a diagnostic algorithm for
ex vivo classification of benign and malignant breast tissue (normal, fibrocystic change, fibroadenoma, and infiltrating carcinoma) with a sensitivity
of 94% and specificity of 96% (N = 58 patients).
gested that Raman spectroscopymay have the utility to guide tissue excision
in vivo during breast cancer surgery with very high sensitivity and specificity to identify carcinoma (100%) and overall accuracy of 93.3%.
subsequent prospective study used Raman spectroscopy to image 129 sites
on breast tissue specimens excised from 21 patients and demonstrated a
sensitivity and specificity of 83% and 93%, respectively, in distinguishing malignant tissue from normal and benign tissue.
technology, spatially offset Raman spectroscopy (SORS), uses multiple
source–detector offsets to achieve greater depth of penetration into tissue
3.25
The resulting Raman
25
Haka et al. found that
25
The authors also sug-
27
A variation of this
26
A
(a)
(b)
(c)
Ex Vivo
Applications 315
Figure 3. Raman spectroscopy measures molecular vibrations of tissue components to produce
chemical and morphologic Raman spectra in (a) normal breast tissue, (b) fibrocystic change, and (c)
invasivecarcinoma. H&E stained sections are shown on the right. Image reprinted with permission.
25
up to 2 mm. Using a SORS probe, Keller et al. classified margins as “positive” or “negative” in 35 breast tissue samples with 95% sensitivity and
100% specificity.
28
Optical coherence tomography
Optical coherence tomography (OCT) has also been proposed as an intraoperative imaging a djunct for ex vivo breast margin assessment. OCT uses
infrared radiation to penetrate tissue up to several millimeters in depth
depending on the tissue type and measures optical scatter from reflected
light using low-coherence interferometry.
images appear similar to ultrasound but with a higher resolution of 10–
20 µm. Using OCT, Nguyen et al. imaged lumpectomy specimens from 37
29
The resultant cross-sectional
316 D. R. Fels Elliott & A. M. Amacher
Figure 4. Optical coherence tomography uses infrared radiation to penetrate tissue. Irregular texture
and shadowing (arrows) are seen in ductal carcinoma in situ (DCIS) and invasive carcinoma (IDC).
Image reprinted with permission.
31
patients undergoing BCS (divided into a training set and feasibility study)
with diagnoses of in situ or invasive carcinoma subtypes and one case of
30
atypical ductal hyperplasia.
The training set (N = 17) established OCT
imaging criteria with areas of higher scatter and heterogeneity indicative
of malignant tissue, while lower scatter areas correlated with normal breast
tissue adipocytes. The acquisition and processing time was approximately
5 s per image with 10–20 parallel images obtained in parallel over a 1× 1cm
area to a depth of 2 mm. The feasibility study (N = 20) identified nine
true positive margins and nine true negative margins, with two false positive results, yielding a sensitivity of 100% and specificity of 82%. Subsequently, a multicenter, prospective study evaluated BCS margins in 46
patients withearly-stage breast cancer using a handheld OCT probe coupled
to an interferometric synthetic aperture microscope (ISAM) image proces-
31
sor.
Cavity-shaved margins were examined ex vivo with the OCT-ISAM
device and yielded rapid, high-resolution images with a tissue penetration
depth of 2–3 mm (Figure 4). Three physicians scored the OCT images and
Ex Vivo
Applications 317
5 of 8 (63%) positive margins (0 mm) were correctly identified, suggesting
potential benefits to patients through minimizing the number of reoperation
procedures. Zhou et al. proposed a multimodal imaging system that combines OCT with confocal microscopy to improve image resolution of the
tissue surface (2µm) while retaining the depth of penetration achieved by
32
OCT.
The 3D images produced from OCT/confocal microscopy showed
distinctive patterns that closely resembled H&E histology for adipose tissue, fibrous stroma, benign lobulesand ducts, cysts, and in situ and invasive
carcinoma (N = 44 specimens, 22 patients). In a similar study, the same
authors applied this multimodal technology to image 35 renal specimens
( N = 19 patients) and diagnose tumor versus normal kidney with high
sensitivity and specificity (88–100% for three observers, kappa statistic
33
0.82).
Applications in Mohs micrographic surgery
Intraoperative frozen section analysis is routinely performed on Mohs
micrographic surgery specimens to assess for margin involvement by nonmelanoma skin cancers. Mohs surgery specimens are very thin and require
a fairly superficial evaluation of the surface for margin positivity. High
resolution is necessary to distinguish between benign skin structures and
tumors, since germinative hair follicles and induction of the epidermis can
closely resemble some non-melanoma skin cancer subtypes.
Rapid lump examination
A simple alternative to frozen section processing that has been described
is rapid lump examination (RLE), a technique that can evaluate thick skin
excision specimens (lumps) with minimal histological processing. In this
technique, specimens are sectioned into 2–4 mm slices, stained with toluidine blue dye, and then placed directly onto a glass slide.
of the specimen is imaged using either digital or stereo microscopy with
polarized light to minimize reflection from the wet surface. RLE does not
destroy or alter skin tissue, so routine FFPE histologic processing can be
carried out following RLE assessment for comparison. Using this technique, Moehrle et al. examined 129 BCC excision specimens by digital
34
The surface
318 D. R. Fels Elliott & A. M. Amacher
microscopy and 78 by stereo microscopy with a resultant sensitivity and
specificity of 91% and 90%, respectively, for digital microscopy and 90%
and 94%, respectively, for stereo microscopy.
34
The authors also developed
a 7-min staining protocol using BerEP4 antibody to highlight BCC tumor
cells, although notably the antibody also stains benign adnexal structures.
Confocal microscopy
Confocal laser scanning microscopy (CLSM) has been proposed as a rapid
intraoperative method to provide real-time high-resolution images of Mohs
surgery specimens. CLSM acquires high-resolution images with a small
surface area (750 × 750 µm) that may be stitched together to produce a
larger mosaic image within minutes and provides a 12 mm field of view
corresponding to 2× magnification on a light microscope.
showed that mosaicing CLSM has high sensitivity (96.6%) and specificity
(89.2%) for detecting basal cell carcinoma in Mohs surgery specimens
( N = 45 confocal mosaics) evaluated by two Mohs surgeons of differing
36
experience levels.
Interpretation of CLSM relies on the refractive index
between tissue types, and techniques have been developed to augment the
contrast between tumor nuclei and the surrounding dermis. Reflectancemode confocal microscopy (RCM) uses acetic acid or citric acid applied
directly to the tissue for 30 s to 5 min prior to obtaining images.
ilarly, fluorescence confocal microscopy (FCM) uses an acridine orange
contrast agent to highlight cell nuclei. Since RCM and FCM modalities
do not permanently alter the tissue, the specimens can later be processed
for either frozen section analysis or routine histologic evaluation, and the
results compared as a gold standard.
Studies using RCM have discovered technical challenges in distinguishing small or ill-defined tumor nests in non-melanoma skin cancers.
For instance, Chung et al. imaged 115 Mohs surgery specimens (92 basal
cell carcinoma (BCC) and 23 squamous cell carcinoma (SCC)) using RCM
with acetic acid and showed that large residual nests of BCC were clearly
identified, while tiny tumor foci were not reliably detected.
Patel et al. found that nodular, micronodular, and superficial BCC subtypes
were well detected, while infiltrative and sclerosing BCC subtypes were
challenging to distinguish from the dermis.
37
A single-center prospective
35
Gareau et al.
37
38
Similarly,
Sim-
Ex Vivo
Applications 319
trial compared the sensitivity RCM with acetic acid to standard FFPE histologic processing for 72 consecutive BCC excision specimens.
39
RCM
images were evaluated by the surgeon within 7.5 min, and the sensitivity
for detection of BCC ranged from 73.7% for lateral margins to 94% in midsections. Although it saved time, RCM lacked the ability to detect small
nests or cords of infiltrative tumor cells in comparison to standard FFPE
histology.
In comparison to RCM, studies using FCM have shown improved
capability to identify tumor in Mohs excision specimens. For example,
Karen et al. showed high sensitivity (96.6%) and specificity (89.2%) in
identifying BCC in 145 Mohs surgery specimens,
concurrent results using CLSM.
36
Subsequently, Bennassar et al. proposed
40
which is comparable to
criteria to interpret FCM images of BCC and differentiate it from cutaneous
appendages, including peripheral palisading, clefting, presence of stroma,
nuclear pleomorphism, nuclear crowding, N/C ratio, demarcation, and fluorescence with a relatively high interobserver agreement (kappa statistic
35
0.68–0.90, two independent observers).
The a uthors showed that a Mohs
surgeon could use these FCM criteria to classify BCC into three major subtypes (superficial, nodular, and infiltrative) with strong histopathological
correlation to H&E slides examined by a dermatopathologist (kappa statistic 0.9; N = 65 cases). Similarly, Longo et al. reported a distinct FCM
appearance for different BCC subtypes, including large tumor islands in
nodular BCC (18/25 cases), small round nests in micronodular BCC (7/7
cases) and small cords in infiltrative BCC (24/30 cases).
41
Overall there
was a strong correlation between FCM and histologic findings for BCC
subtypes (kappa statistic 0.9, N = 64 cases), but sebaceous glands and
intense stromal reaction were identified as potential confounders in six
cases.
Further advancements to confocal microscopy have been proposed
to maximize potential clinical utility in the Mohs surgery setting. Abeytunge et al. developed a technique called “strip mosaicing” to reduce the
time required to produce a confocal mosaic from 9 to 3 min (approximately 25 × 25 mm of tissue) by acquiring strips of images with an aspect
42–44
ratio of 10:1, which is greater than the standard 1:1 ratio.
Additionally, multimodal confocal microscopes have been developed using FCM
to highlight cell nuclei combined with RCM to highlight cell cytoplasm