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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5511_Библиотеки_им_академика_М_И_Перельмана.pdf
cervical cancer cases (n = 2855) indicates that although frequencies
varied, HPV 16 followed by HPV 18, were most commonly detected
in Africa, Central-South America, South Asia, and North America.
HPVs 45, 31, and 33 were also seen globally.
7
There are many different immunologically distinct types of HPVs,
which have been linked to specific conditions: genital cancer (HPVs
16, 18); genital warts (HPVs 6, 11); epidermodysplasia verruciformis
EV (HPVs 5, 8); and cutaneous warts (HPVs 1, 2). HPV 16 and
HPV 18 appear to be most frequent in cases of high-grade squamous
intraepithelial lesions (HSIL) or cases of invasive squamous cell carcinoma (SCC), although the relative incidence of different types varies
in different regions.
8
Infection does not inevitably lead to pathologi-
cal changes and regression occurs in many cases.
HPV DNA testing may be used clinically for (a) screening, either
alone or as an adjunct to cytology; (b) triage of patients with uncertain
Pap results; (c) monitoring patients post-treatment. Amplification of
viral DNA may be through target amplification (PCR) or signal amplification (Hybrid Capture II). HPV testing in combination with Pap
248 M Wu
Fig. 9 A biphasic tumor cell population including epithelial tumor cells and stromal type of tumor cells. This shows example of recurrent MMMT seen in Pap test.
Pap stained ThinPrep slide, original magnification 100×.
cytology has improved negative predictive value (approaches 100%).
Such combination results in fewer unnecessary colposcopies. A metaanalysis to assess the accuracy of HPV DNA testing as an alternative to
repeat cytology in women who had equivocal results on a previous Pap
smear suggests that HC II assay has higher sensitivity and similar specificity compared to the repeat Pap smear (ASCUS as threshold) for CIN 2+
among women in this patient group.
9
However, it is still controversial
as to whether HPV testing would be able to replace the Pap testing.
Other efforts aimed to detect precancerous cervical lesions using
newly developed rapid biochemical tests that are affordable, accurate, simple to use, and appropriate for low-resource settings are
being developed, namely batch test and rapid strip test. The batch
test developed by Digene Corporation, US, will use an instant
photo signal output. Images of 46 samples will be compared visually on a film with positive and negative controls. The results can be
obtained in about two hours. It is suitable for a small clinic or
mobile unit. The rapid strip test promises to discriminate between
neoplastic transformation and infection by HPV. Arbor Vita
Corporation technology detects a biomarker (E6 oncoprotein)
which correlates with the neoplastic transformation of cells and cervical cancer. The result may appear in 20 minutes. The ELISA prototype is now being adapted to an immuno-chromatographic strip
format capable of detecting common high-risk HPV types. Efforts
are focused on improving sensitivity.
Based on a review article written by Cox JT in 2006,
10
HPV tests
in combination with Pap tests are 96% to 100% sensitive for detection
of cervical intraepithelial neoplasia (CIN 2 or CIN 3) and cancer.
However, because HPV infection is common in young women and
most commonly transient, the 2006 consensus guidelines were that
1. HPV testing is not recommended as part of primary cervical
screening for women younger than 30 years of age.
2. HPV testing is recommended for women of any age for the clar-
ification of ASCUS and as an option for follow-up of women
with HPV-positive ASCUS, ASC-H, and or LSIL not found to
have CIN 2 or CIN 3.
Cytology for Early Diagnosis of Gynecologic Cancer 249
3. HPV testing is also recommended as an alternative to colposcopy
and/or cytology for follow-up of treated cases.
Proper use of HPV testing improves the management of
women with cytological abnormalities. In addition, a negative
HPV test in combination with a normal Pap test result in women
age 30 and older allows the safe extension of the interval between
cervical screenings. Thus, when used properly, HPV testing may
reduce morbidity and mortality and does so in a cost-effective
manner. Presently, routine Pap screening test in combination with
reflex high-risk HPV testing becomes a standard practice in the
US. Many precancerous lesions are detected and treated in very
early stages in patients routinely participating in Pap screening programs. Hopefully, high-risk HPV vaccines will break the cycle even
earlier.
HPV virus is not highly immunogenic, regression of HPV being
dependent on the immune response, occurring less frequently in
immunocompromised patients such as renal transplant recipients.
Details of this immune response are not completely understood, but
humoral, cellular, and innate immunity may all play a role.
Quadrivalent HPVs 6, 11, 16, 18 vaccine (GARDASIL) comprises virus-like particles produced in recombinant yeast.
11
The vaccine is adsorbed on the Merck proprietary aluminum adjuvant, which
strengthens its immunogenicity.
HPV 16/18 vaccine is a prophylactic candidate that contains
bivalent viral-like protein particles, L1, adjuvanted by AS04 as an
enhancing agent.
12
This vaccine was designed to target HPV types 16
and 18.
CerVax 16 is an experimental therapeutic product which uses
E6/E7 proteins (identical to those of HPV16) and a quillaia saponinbased adjuvant capable of promoting both humoral and cell-mediated
immunity.
13
It was recently reported
12,14
that clinical trials have documented
the safety, efficacy, and immunogenicity of the quadrivalent and bivalent HPV L1 virus-like particle vaccines. These vaccines have demonstrated greater than 90% efficacy in preventing HPV-related
250 M Wu
neoplasias of the lower genital tract. The quadrivalent HPV vaccine
has been found to be more than 95% efficacious in preventing genital
warts. These vaccines have been shown to protect against a wider
range of oncogenic HPV types. Nonetheless, women must continue
to have routine cervical cytological screening.
Molecular Studies
A number of epigenetic alterations occur during all stages of cervical carcinogenesis in both human papillomavirus and host cellular
genomes, which include global DNA hypomethylation, hypermetylation of key tumor suppressor genes, and histone modifications.
The reversible nature of epigenetic changes constitutes a target for
transcriptional therapies, namely DNA methylation and histone
deacetylase inhibitors. To date, studies in patients with cervical
cancer have demonstrated the feasibility of reactivating the expression of hypermethylated and silenced tumor suppressor genes as
well as the hyperacetylating and inhibitory effect upon histone
deacetylase activity in tumor tissues after treatment with demethylating and histone deacetylase inhibitors. In addition, detection of
epigenetic changes in cytological smears, serum DNA, and peripheral blood are of potential interest for the development of novel
biomolecular markers for early detection, prediction of response,
and prognosis.
The realization that genetic and epigenetic alterations are present
at the earliest steps of the malignant progression of cervix uteri has led
to testing the presence of these abnormalities, such as p16
expression.
15,16
Overexpression of the protein p16
Ink4a
is typical for the
dysplastic and neoplastic epithelium of the cervix. However p16
Ink4a
negative CINs and carcinomas do exist. All stages of CINs and
carcinomas analyzed are heterogeneous with respect to
p16
Ink4a
expression. So p16
Ink4a
negativity is not a sufficient reason to
exclude a patient from the high risk group. Since normal cervical
epithelium is negative for p16
Ink4a
, the ratio p16
Ink4a
positive/p16
Ink4a
negative samples should increase as the severity of the lesion progresses. Application of immunohisto-/cytochemical test for p16
Ink4a
Cytology for Early Diagnosis of Gynecologic Cancer 251
may be regarded as a supplementary test for the early diagnosing of
cervical cancer.
A large number of studies looking at the methylation status of
tumor suppressor genes have uncovered that some genes are found
hypermethylated in preinvasive lesions, raising the possibility that
testing for methylation of either of these or of a set of these may prove
to be a useful screening tool.
17–21
However, there is limited information with respect to the sensitivity and specificity of methylated genes
for the identification of women with cervical dysplasia and cancer as
well as comparisons of results using different sources of samples,
either exfoliated cells or paraffin-embedded biopsy samples. In this
regard, a very comprehensive study
22
investigated the methylation
profile of 20 genes (p16, p15, CCND2, RASSF1, RARb, TWIST1,
SYK, HIC1, VHL, PRDM2, SFN, MLH1, MGMT, APC, CDH1,
and CDH13) in exfoliates and biopsies of 319 women that participated in a cytology screening study. By logistic regression, the authors
determined the best set of candidate genes for employment as disease
markers. The key findings are (1) similar detection rate of methylation
regardless of sample source; (2) CDH13, DAPK1, RARb, and
TWIST1 were the genes showing a statistically significant increase
with lesion severity and DAPK1, RARb, and TWIST1, the best panel
of hypermethylated genes; (3) at least one of the three genes was
hypermethylated in 57% of samples with CIN 3/CIS and in 74% with
invasive cancer, but in only 5% of samples with CIN 1; (4) estimated
specificity of the panel was 95% with sensitivity of 74% (95% confidence interval CI 95%, 73%–75%) for invasive carcinoma and 52%
(95%, 49%–55%) for CIN 3/CIS. These findings provide preliminary
evidence on the potential usefulness of a panel of genes to be tested
for hypermethylation in cytology samples; however, additional studies
are needed before this epigenetic-based screening test could be
adopted. The methylation status of several genes present in the serum
or plasma of patients with cervical cancer has been studied with regard
to their prognostic significance.
23–25
Together, these data encourage
further studies to find a set of methylated genes that would have
prognostic significance but that would also serve as surrogate markers of efficacy of epigenetic therapies.
252 M Wu
Circulating nucleic acids represent a biomarker that might be
used in early detection of cancer, in the follow-up of patients with
cancer or as a prognostic factor. Presence of nucleic acids in plasma or
serum of patients with cancer has been recognized.
26–32
One study
32
analyzed the effect of tumor and chemotherapy upon the levels of
nucleosomes in vitro, in vivo and in cervical cancer patients and
revealed that (1) most of circulating nucleosomes originate from the
tumor and that chemotherapy produces an early rise most likely due
to tumor apoptosis; (2) nucleosomes are rapidly cleared from circulation; (3) chemotherapy within the therapeutic range of doses has no
effect on nucleosome levels in healthy mice and rats. This data suggests that the determination of circulating nucleosomes pre- and posttreatment could be a useful test to predict response to chemotherapy
in cancer patients.
ENDOMETRIAL NEOPLASIA
Endometrial Cytology
Endometrial cells are generally detected through Pap tests since
direct endometrial sampling is rarely done. However, only the type
of endometrial carcinoma that is easily shed from superficial
endometrium can be detected by Pap test. Early cytological detection
of endometrial carcinoma may be improved by vigorous follow-up of
patients with high risk signs or symptoms such as postmenopausal
bleeding and direct endometrial sampling whenever an atypical
endometrioid glandular cell type is noted on Pap test.
Endometrial glandular cells, stromal cells or both can be identified in a Pap test. Normal endometrial cells can be seen in the first half
of the menstrual cycle. Their presence in the second half of the cycle,
particularly in women older than 40 years of age or postmenopausal,
is considered to be abnormal or a risk factor for endometrial hyperplasia or neoplasia. Especially, when the nucleus is atypical (larger than
squamous intermediate cell nucleus and with nucleolus), suspicion for
endometrial carcinoma should be higher. Shedding of abnormal or
atypical endometrial cells is always abnormal, regardless of its relationship to the menstrual cycle.
Cytology for Early Diagnosis of Gynecologic Cancer 253
Updated Endometrial Carcinogenesis and Molecular Studies
It is generally accepted that there are two types of endometrial
carcinomas:
Type I, estrogen dependent, well differentiated endometrial carcinomas.
Type II, ovarian serous adenocarcinoma-like, poorly differentiated type.
In recent decades, progress has been made in defining endometrial precancers. Endometrial intraepithelial neoplasia has been widely
accepted as a precancer of type I endometrial cancer, while endometrial glandular dysplasia (endometrial intraepithelial carcinoma, EIC)
is a newly described entity as a probable precancer of type II cancer.
33
Biomarkers of phosphatase and tensin homolog (PTEN), β-
catenin, p53, Insulin-like growth factor II mRNA-binding protein 3
(IMP3), N-myc Downstream-regulated Gene 1 (NDRG1) are help-
ful for the early detection of endometrial glandular dysplasia as well as
for type II endometrial cancers.
33–37
254 M Wu
Fig. 10 Endometrial adenocarcinoma appears in cervical Pap test in a flower-like
glandular pattern. Pap stained ThinPrep slide, original magnification 100×.
Based on Boruban et al.,38biomarkers whose expression is altered
in cases of endometrial hyperplasia or cancer such as progesterone
receptors, insulin-like growth factor I, retinaldehyde dehydrogenase
type II, and secreted frizzled-related protein 4, seem to be promising
to use as early-stage tumor markers. Mutation of PTEN is present in
83% of endometrial adenocarcinoma cases, making it the most frequent early molecular genetic alteration in type I endometrial tumors,
which are generally associated with hyperplasia. p53 gene mutation is
not found in endometrial hyperplasia, but researchers have detected
this mutation in 20% of cases of endometrial carcinoma and 90% of
cases of serous endometrial tumors. Cyclooxygenase-2 is important in
tumorigenic transformation of hyperplasia. Expression of cyclooxygenase-2 decreases apoptosis, increases angiogenesis, and is related to
invasiveness. Cyclooxygenase-2 expression increases significantly in
cases of well-differentiated endometrial adenocarcinoma.
Hepsin is a type II transmembrane serine protease originally identified in the human liver as a cDNA clone. Hepsin was found to be
significantly overexpressed in cancer samples compared to matched
various tissues such as prostate, renal, ovarian carcinoma. Matsuo
et al.
39
have examined Hepsin expression and its clinicopathological
significance in endometrial cancer. The authors have found that
(1) Hepsin expression was significantly higher in endometrial cancer
compared to normal endometrium and endometrial hyperplasia;
(2) high levels of Hepsin expression were associated with advanced
stage (p < 0.001), high grade (p = 0.002), depth of myometrial invasion (p < 0.001), cervical involvement (p = 0.007), lymph node
metastasis (p = 0.001), lympho vascular space (LVS) involvement
(p = 0.006), ovarian metastasis (p = 0.002), and peritoneal cytology
(p = 0.03) of endometrial cancer. They have concluded that Hepsin
protein expression could be an important indication for increased risk
for endometrial cancer.
Antibodies to the factor HLDF
40
are shown to be specific
markers of apoptosis and permit the estimation of the rate of programmed cell death in the course of a normal menstrual cycle and in
pathologic endometrial processes. HLDF expression in the epitheliocytic
cytoplasm makes it possible to evaluate apoptosis at early stages, before
Cytology for Early Diagnosis of Gynecologic Cancer 255
the emergence of the first morphological signs and after apoptotic
body formation. The study shows increased apoptotic processes at the
end of a normal menstrual cycle and during neoplastic cell transformation. Antibodies to the HLDF factor may be used as a new
immunohistochemical marker for the differential diagnosis of benign
and malignant endometrial processes.
Using color texture analysis, Neofytou et al.
41
developed a system
for the classification of hysteroscopy images of the endometrium for
the early detection of gynecological cancer.
OVARIAN NEOPLASIA
Ovarian and Peritoneal Cytology
Ovarian cancer is the most lethal gynecological cancer. It can arise
from any cell type of ovary, including surface epithelial cells, germ
256 M Wu
Fig. 11 This is a picture taken from the same patient as that in Fig. 10 with a
exception that this slide was process by SurePath preparation. Comparing to the well
preserved glandular pattern seen in ThinPrep slide, although the individual nuclear
details are clearly visualized, the architecture or tumor seems to be disrupted. Pap
stained SurPath slide, original magnification 100×.
cells, granulosa or stromal cells. However, the majority of ovarian cancers arise from the surface epithelium, a single layer of cells that cover
the surface of ovary. The lack of a reliable and specific method for the
early detection of epithelial ovarian cancer results in diagnosis occurring most commonly at late clinical stages, when treatment is less
effective.
Cytology can be used as a tool to detect ovarian cancer by analyzing or examining (1) ovarian cyst fluids via fine needle aspiration;
(2) ascites fluid via paracentesis; (3) pre- and post-surgical peritoneal
washings.
Benign ovarian cyst fluid generally has low cellularity. An ovarian cyst
fluid specimen usually contains foamy macrophages, hemosiderinladen macrophages in addition to its corresponding benign cyst lining
cells such as
1. granulosa cells in follicular cyst;
2. luteinized granulosa cells in luteal cyst;
3. cuboidal or flat serous cells in serous cyst;
4. columnar or flat mucinous cells in mucinous cystadenoma;
5. endometrial cells in endometriosis;
6. squamous cells/anucleated squames in dermoid cyst or mature
teratoma.
Malignant counterparts of ovarian cystic lesions would reveal cells with
some cytoplasmic features of their benign counterparts but contain
malignant nuclei and with high N/C ratios.
Borderline or atypical cystic lesions contain cells falling into the category in between benign and malignant types.
It is generally not too difficult to differentiate clearly benign from
definitely malignant cells. It is the borderline or atypical cells that are
often not only difficult to deal with both in cytology sign-out and
clinical management, but also very important in early detection of
ovarian cancers. This author would propose that efforts should be
Cytology for Early Diagnosis of Gynecologic Cancer 257