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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
250 J. Mirkovic & E. Yang
(a) (b) (c)
(d) (e) (f)
Figure 1. Spectroscopy parameter maps acquired by quantitative spectroscopic imaging (QSI) overlaid on a white-light photograph of a cervix. (a) A, reduced scattering coefficient at 700 nm (mm
(b) B, wavelength dependence of reduced scattering coefficient. (c) [Hb], total hemoglobin concentration (mg/mL). (d) α, oxygen saturation (%). (e) Coll, collagen concentration (a.u.). (f) NADH,
concentration of reduced form of nicotinamide adenine dinucleotide (a.u.). Pixels with no spectroscopy
data due to the presence of specular reflection, motion, acute tissue angle relative to QSI system, or
non-cervical substances are uncolored in the parameter maps. Note that specular reflection observed
in the white-light photographs is not necessarily the sites of specular reflection for spectroscopy measurements. A portion of the speculum (bottom of photograph) is visible.
Source: Reprinted with permission from Lau et al.
11
−1
it distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this study agree with those of an earlier contact-probe
14
study,
validating the robustness of QSI for identifying HSIL. Figure 1
shows spectroscopy parameter maps as acquired by QSI but superimposed
on a white-light photograph of a cervix.
Confocal microscopy
Confocal microscopy is an optical technique developed in the 1950s to
overcome the poor contrast inherent in imaging thick specimens with a
conventional microscope. In contrast to conventional optical microscopy,
).
Gynecologic Tract 251
confocal microscopy allows for the elimination of out-of-focus light and for
the collection of serial optical sections from thick specimens. A confocal
microscope can be implemented in both transmission and reflection modes.
Both elasticscattering and fluorescencecan be used as a source of contrast in
confocal microscopy. Thus, this technique can provide non-invasive highresolution structural and biochemical imaging at the cellularlevel at varying
depths in the cervical epithelium.
Several studies explored the potential of reflectance and fluorescence
confocal microscopy to image the morphologic and biochemical changes
associated with CIN and cancer.
54–56
Increased epithelial fluorescence,
attributed to mitochondrial NADH, as well as decreased collagen fluorescence, attributed to collagen matrix degradation by matrix metalloproteinases, have been observed for dysplastic compared to normal cervical
57
freshly excised tissue using fluorescence confocal microscopy.
Nuclearto-cytoplasmic ratio, nuclear density, and scattering coefficient were established as biomarkers associated with the progression of CIN.
54,56, 58
One
study demonstrated variability within and the subtle differences between
the normal columnar epithelium, normal squamous epithelium, CIN, and
stromal tissue in the context of fluorescence confocal endomicroscopic
in vivo pathology.
59
In vivo reflectance confocal microscopy was successful in visualizing
the increase in nuclear-to-cytoplasmic ratio and nuclear density associated
with the progression of CIN. A fiber-optic reflectance confocal microscope
(FRCM) was used for in vivo imaging of cervix.
60,61
These studies demonstrated the minimal change of nucleus-to-cytoplasm ratio from the basal
layer to the upper epithelial layers in dysplastic epithelium, but detected a
significant increase of the nucleus-to-cytoplasm ratio with scanning depth
in normal epithelium. A feasibility in vivo study of 18 patients undergoing colposcopic examination of the cervix using FCRM showed that this
technique can be used to image epithelial tissue with subcellular resolution
in vivo.
61
Confocal fluorescence endomicroscopy for in vivo microscopic
imaging of cellular structures during colposcopy was used in a study by
Tan et al.
58
Normal and abnormal regions of the cervix were imaged
following topical application of a fluorescent dye (acriflavine). CIN was
252 J. Mirkovic & E. Yang
Table 1. Confocal image pattern classification to predict cervical pathology.
Grading Cellular architecture
O (Normal) Regularly spaced cell nuclei measuring 4–12μm in diameter, which are
spaced approximately 10–30μmapart
1 (CIN1) A slight increase in cellular density per field of view (compared to
normal) and irregularly spaced cell nuclei and slight nuclear atypia.
2 (CIN2) Enlarged (10–15μm) cellular nuclei that are densely concentrated,
irregularly spaced and stain strongly with the fluorescent contrast agent
3 (CIN3) Extensive cellular proliferation and cellular atypia with nuclear
fluorescent staining (hyperchromasia) and nuclear pleomorphism
Source: Reprinted with permission from Tan et al.
58
characterizedby an increase in nuclear density, size, and cellular atypia. The
sensitivity for detection of CIN was 97%. The specificity for predicting normal epithelium-CIN1 was 80% and 93% for CIN2-CIN3. Table 1 outlines
the confocal image pattern classification used in this study to predict cervical pathology. Figures 2 and 3 show cervical epithelium via colposcopy,
confocal endomicroscopy, and routine histology.
These results demonstrate the potential of confocal microscopy to
identify the morphological and biochemical changes associated with the
progression of precancer, especially if quantitative, rather than qualitative
assessment of images would be developed.
Optical coherence tomography
OCT is an optical technique developed in the early 1990s for non-invasive
cross-sectional imaging in biological systems. OCT uses low-coherence
interferometry to produce a two-dimensional image of elastic light scattering from tissue in a way analogous to ultrasound imaging. In the most basic
form, an OCT instrument consists of a Michelson-type interferometer with
a focused sample arm beam and a lateral-scanning mechanism. OCT has
longitudinal and lateral spatial resolutions of a few micrometers, a depth
of penetration of up to 2 mm, detects reflected signals as small as 10
−10
(a)
(c)
(b)
d)
Figure 2. Features of the cervical epithelium examined using (i) colposcopy, (ii) confocal endomicroscopy, and (iii) conventional histology (H&E staining).
(a) Normal cervix. Colposcopy showed metaplasia at 6 o’clock while confocal imaging and histology showed uniform arrangement of glycogenated cells
through the full thickness of the squamous epithelium. (b) CIN1. Confocal imaging of a small area of low-grade acetowhite epithelium at 1 o’clock showed
slight nuclear atypia. Histology confirmed the presence of CIN1 in the basal zones of the epithelium, which was frequently associated with HPV infection.
(c) CIN2. Colposcopy showed ectopy with a large area of acetowhite epithelium at 12 o’clock. Confocal imaging showed moderate dysplastic changes
in the squamous epithelium. Histology showed koilocytosis (indicating HPV infection) associated with nuclear size variation and disorderly maturation of
squamous cells in the basal two-thirds of the epithelium. (d) CIN3. Colposcopy showed acetowhite epithelium at 12 o’clock. Examination with confocal
imaging showed significant variation in nuclear size and shape and a strong fluorescent staining intensity. Histology was characterized by near full-thickness
loss of maturation of squamous cells associated with nuclear pleomorphism and intraepithelial mitoses.
∗
Note:
confocal image site. Bars = 100μm.
Source: Reprinted with permission from Tan et al.
58
Gynecologic Tract 253
254 J. Mirkovic & E. Yang
(a)
(b)
(c)
Figure 3. Morphological features of the normal cervix examined using (i) colposocopy, (ii) confocal
endomicroscopy, and (iii) conventional histology (H&E staining). (a) Dermal papillae of the squamous
epithelium appeared as small swirl-like aggregates of cells using confocal imaging. In conventional
histology, the tangential cut showed the elongated stromal papillae containing numerous capillaries.
(b) Endocervical glands could be imaged in a cervix with ectopy as areas of br ight fluorescence
defined by distinct borders. Histology of the glands showed a well-defined single layer of columnar
cells lining the glands. (c) The SCJ was imaged using confocal endomicroscopy as a distinct line of
different fluorescent staining patterns separating the glycogenated cells of the squamous epithelium
and the connective tissue matrix of the columnar epithelium. Conventional histology showed the
characteristic step-like feature of the SCJ defined by the difference in hei ghts of the squamous and
glandular columnar epithelia.
∗
Note:
confocal image sites. Bars = 100μm.
Source: Reprinted with permission from Tan et al.
58
Gynecologic Tract 255
of the incident optical power, and allows for non-invasive visualization of
tissue in real time.
OCT has become part of standard of care in ophthalmology but
has also found clinical applications in the fields of gastroenterology
and dermatology. In recent years, promising advances have also been made
in the detection of cervical cancer and its precursor lesions with OCT.
62–68
In vivo pilot studies have shown that in premenopausal women, average
OCT reflection intensities from abnormal cervical epithelium were signif-
63,68
icantly stronger than those from the normal epithelium.
These differences were attributed to neoplastic changes in the chromatin texture and
the nuclear morphology and texture.
A combination of OCT and colposcopy may improve the detection of
HSIL. The diagnostic efficacy of real-time in vivo OCT as an adjunct to colposcopy was evaluated in the study of 299 women. This study demonstrated
that by adding OCT to colposcopy, the specificity for CIN-2 and higher
(CIN2+) increased from 83% to 93%, but the sensitivity decreased.
66
A system that integrated an OCT device into a microscope was used to
collect and analyze 160 images from 20 loop electrosurgical excision procedure (LEEP) specimens and showed 84–88% sensitivity and 65–69%
specificity for detecting high-grade squamous intraepithelial lesions (HSIL;
CIN2/3).
64
Incorporation of epithelial brightness in the algorithm for the detection of cervical neoplasia/cancer may improve the diagnostic accuracy
of OCT. One study evaluated OCT epithelial brightness in 476 women
with abnormal cervical cytology and/or HPV-positive status undergoing
colposcopic evaluation/unaided visual inspection and OCT-matched cervical biopsies. Mean OCT image brightness differed significantly between
each preinvasive histological grade and invasive cancer (p <.01 for all
comparisons).
62
Polarization-sensitive OCT (PS-OCT) technique was used to quantify
the polarization changes caused by the scattering changes induced by CIN.
A total of 71 images from 18 patients undergoing cervical conization were
examined and compared to histologic findings to show 94.7% and 71.2%
sensitivity and specificity.
65
256 J. Mirkovic & E. Yang
IVM detection of cervical neoplasia in resource-poor setting
In developing countries without resources to support Pap smear cytology
and colposcopy, cost-effective approaches which enable single-visit “seeand-treat” protocols offer the potential to reduce morbidity and mortality due to this preventable disease. Pilot clinical studies suggest that the
evaluation of suspicious lesions by IVM may assist in ruling out immediate cryotherapy, thus increasing the efficiency of current “see-and-treat”
programs.
63,67, 69–72
The efficacy of real-time in vivo OCT as an adjunct to unaided visual
inspection with acetic acid (VIA) for diagnosis of CIN2+ was evaluated
in a prospective cross-sectional comparative trial including 183 women in
a resource-poor setting. For VIA alone, the sensitivity and specificity in
detecting lesions greater than or equal to CIN2 were 43% and 96%. With
the addition of OCT, the sensitivity increased to 62%, but the specificity
decreased to 80%.
67
Vulva
Histopathologic overview
The externally visible portion of the vulva is lined by squamous epithelium, which is non-keratinized within the vulvar vestibule and keratinized
elsewhere. Major and minor vestibular glands and paraurethral glands may
be found within the s ubepithelial stromal tissue with connections to the
squamous epithelium. The most common (over 90%) and readily evaluable lesions of the vulva are those of squamous origin, including in situ and
invasive squamous cell carcinomaof HPV-associated andHPV-independent
etiologies. Of note, HPV-associated vulvar intraepithelial neoplasia (usual
or classic VIN) is histopathologically indistinguishable from those arising
in other sites of the lower anogenital tract, including the vagina, cervix,
anus, and penis.
ferentiated vulvar intraepithelial neoplasia, dVIN) is morphologically distinct from usual VIN and may arise in association with lichen sclerosus
in an older population. These premalignant lesions tend to give rise to
73
HPV-independent vulvar intraepithelial neoplasia (dif-
Gynecologic Tract 257
keratinizing squamous cell carcinoma and share morphologic features with
other HPV-independent squamous dysplasia/carcinomas (e.g. skin, oral
cavity, and larynx). Other less common epithelial malignancies include
extramammary Paget disease, malignant melanoma, basal cell carcinoma,
and Bartholin’s gland carcinoma. A variety of non-infectious vulvar dermatoses, infectious diseases, as well as mesenchymal neoplasms may also
arise in this site. The literature on IVM application in the vulva focuses on
the detection of neoplastic epithelial lesions.
IVM features of normal vulva
Normal vulvar epithelium has been described using OCT and reflectance
confocal microscopy(RCM). OCT visualizesepithelium on cross-sectionat
near-cellular resolution (10–20μm) and provides predominantly architectural information. The epithelium appears as a dark gray homogeneousband
with a clear visualization of the epithelial,basement membrane, and stromal
74
layers.
showing a honeycomb pattern of the epidermis interrupted by round to
elongated papillary dermal projections rimmed by bright monomorphic
cells.
Rare bright cells that show a dendritic morphology may represent scattered
melanocytes or Langerhans cells.
RCM visualizesen face epithelium at a higher resolution(1–5μm),
75
This brightness is thought to represent pigment-laden basal cells.
IVM features of vulvar pathology
Squamous dysplasia and carcinoma
Wessels et al. have investigated the utility of in vivo OCT for the distinction of vulvar squamous dysplasia and normal epithelium.
report greater epithelial thickness and attenuation coefficients in dysplasia.
They confirm their findings in a subsequent study in which quantitative
analysis of both parameters at the time of surgical excision shows statistically significant differences in (pre)malignant squamous lesions and benign
epithelium (Figures 4 and 5).
74
Both parameters are in keeping with the histologic criteria of dys-
77
plasia.
Increased epithelial thickness corresponds to acanthosis generally
76
The authors
258 J. Mirkovic & E. Yang
(c)
(a)
(b)
Figure 4. Three-dimensional OCT image (a) and 2-dimensional OCT image (b) of an inflammatory
area of the vulva. In (c), the attenuation of the OCT signal of the area marked with the blue bar is
shown and (d) shows corresponding histology (hematoxylin-eosin coloring) of the OCT image.
Note:The“∗” shows the horny layer of the skin, and the “#” shows the epidermal layer.
Source: Reprinted with permission from Wessels et al.
(d)
74
seen in low- and high-grade dysplasia. Increased attenuation coefficient
(light scattering property) of dysplastic epithelium likely corresponds
to increased nuclear-to-cytoplasmic ratios, hyperchromasia, and irregular
nuclear contours.Therefore, although cytologic features ofdysplasia are not
directlyvisualized at the OCT resolution utilized by the authors (10–20μm),
changes in nuclear qualityare detected indirectly through optical manifestations of altered light scattering properties. However, the inability to evaluate
cytologic features leaves us vulnerable to overcalling reactive/inflammatory
changes that may ostensibly appear similar to dysplasia on OCT imaging.
Cytomorphologic features of squamous cell carcinoma have been
extensively evaluated in the skin
78
(see Chapter 4) by RCM and may
be extrapolated to evaluate HPV-independent vulvar squamous dysplasia/carcinoma to some extent. IVM literature specifically describing the
morphology of usual VIN vs. dVIN, along with their invasive counterparts
is limited.
Gynecologic Tract 259
(a)
(b)
Figure 5. Three-dimensional OCT image (a) and 2-dimensional OCT image (b) of VSCC. In (c),
the attenuation of the OCT signal of the area marked with the blue bar is shown and (d) shows
corresponding histology (hematoxylin-eosin coloring) of the OCT image.
Note:The“∗” shows the horny layer of the skin, and the “#” shows the thickened epidermal tissue.
Source: Reprinted with permission from Wessels et al.
(c)
(d)
74
Melanoma
Features of vulvar melanosis and melanoma have been described using
75
RCM.
epithelium as described above except that the cells lining the papillary
projections appear more refractive; this may correspond with greater pigmentation of basal keratinocytes. Cases of vulvar melanoma show disarray
of the normal epithelial architecture characterized by loss of regularity and
definition of dermal papillary projections. In addition, bright, atypical cells
with round, spindled, or dendritic morphology are seen, sometimes completely effacing normal architecture; these cells are thought to represent
malignant melanocytes. RCM may be useful in the diagnosis of melanoma,
assessment of surgical margins, and clinical follow-up.
Vulvar melanosis demonstrates similar features to uninvolved
Basal cell carcinoma
In vivo RCM has been used to identifybasal cell carcinoma of the vulva79by
extrapolating from dermatologic IVM criteria. In vivo imaging has allowed