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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5511_Библиотеки_им_академика_М_И_Перельмана.pdf
There is no recognized model for ovarian cancer screening, nor any
clinical test approved for early-stage diagnosis.
1
To be useful in the clinical setting, a biomarker should be accu-
rate for diagnosis, disease progression monitoring, disease recurrence
prediction and treatment response monitoring. On a practical point
of view, a biomarker should also be sufficiently non-invasive (i.e. performed on body fluid) and inexpensive to allow widespread applicability. Given that prevalence of ovarian cancer, strategies for early
detection must have high sensitivity for early stage disease (> 75%),
but must have extremely high specificity (99.6%) to attain a positive
predictive value of at least 10%.
2
The current ovarian cancer biomarkers have high sensitivity for clinically diagnosed disease, but very few
of them have high sensitivity for early stage disease.
Serum and Urine Biomarkers
Ca 125
3
Ca 125 is the best performing single biological marker for ovarian
cancer. Ca 125, a high molecular weight mucin (MUC 16), was first
detected with a radioimmunoassay in patients with advanced ovarian
cancer. It is approved only for the use of monitoring disease after
treatment. Despite the fact that 80% of women with high stage disease have elevated Ca 125, Ca 125 is elevated in only 50%–60% of
women with early stage disease. However, Ca 125 levels can be elevated 10–21 months prior to conventional diagnosis. Ca 125 is also
elevated in numerous benign ovarian conditions (particularly in a
premenopausal population with endometriosis, adenomyosis and retrograde menstruation) but not on every ovarian neoplasms, thus compromising its sensitivity and specificity. Greater specificity can be achieved
by combining Ca 125 and transvaginal sonography (TVS) and/or by
monitoring Ca 125 over time.
Ca 125 and Transvaginal Sonography (TVS)
Screening modalities evaluated to date have included TVS and the use
of serum tumor marker Ca 125. Using a two-stage strategy, however, a
268 F Penault-Llorca
combination of Ca 125 followed by sonography does attain specificity
in excess of 99.6%. Specificity of Ca 125 could be further improved by
following the trend of Ca 125 with an algorithm that estimates the risk
of ovarian cancer.
An algorithm has been developed to calculate the risk of ovarian
cancer based on serial Ca 125 values and refers patients at highest
risks for TVS. Using this strategy, Jacobs et al.
2
randomized 21,962
postmenopausal women over 45 years of age in the UK to a control
group or to a screened group. Ca 125 was measured annually for
three years. An elevated Ca 125 level > 30 units/mL prompted
that TVS and surgery was undertaken if the TVS demonstrated a
pelvic mass. Among 10,985 women screened, in which 29 operations
were performed to detect six cancers, providing a positive predictive
value of 21%. Median survival in the screened group (72.9 months)
was significantly greater (p = 0.0112) than that in the control group
(41.8 months).
Use of the algorithm is currently being evaluated in a trial with
200,000 women in the UK (accrual has been completed) that will
critically test the ability of a two-stage screening strategy to improve
survival in ovarian cancer. Nevertheless, this approach is still far from
perfect. Use of Ca 125 as an initial stage in a two-step screening strategy is limited by the fact that Ca 125 is expressed by only 80% of
epithelial ovarian cancers. The sensitivity of such an approach is further reduced due to an inability of TVS to detect a mass that has yet
to become appreciable.
4
Ca 125 in Combination with Other Serum or
Urine Markers
1,3,5
Whatever the outcome, additional serum markers will be required to
detect all patients in an initial phase of screening. More than 30 serum
markers have been evaluated alone and in combination with Ca 125.
Recent candidates include: HE4, mesothelin, M-CSF, osteopontin,
kallikrein(s), lysophosphatidic acid (LPA) and soluble EGF receptor.
In a study of 89 sera from patients with stage I ovarian cancer, use of
three markers in combination (Ca 125, OVX1 and M-CSF) detected
Molecular and Biological Diagnosis of Early Gynecologic Cancers 269
84% of cancers, whereas Ca 125 alone detected 69%. Specificity, however, declined from 99% to 84% with the combination. Sensitivity has
been improved by 5%–15%, but specificity has inevitably been reduced.
Mathematical Models
Artificial neural network (ANN) analysis, as a statistical modeling
tool, has demonstrated the ability to assimilate information from multiple sources and detect subtle and complex patterns. An ANN-based
composite diagnostic index using a panel of four serum markers,
Ca 125II, Ca 72-4, Ca 15-3 and lipid-associated sialic acid (LASA),
was evaluated for its ability to discriminate malignant from benign
pelvic masses. Different sets of population (training and validation
sets) i.e. healthy, with benign condition arising from the ovary and
with ovarian cancers (including respectively 27 and 38 stage I cancer)
malignant pelvic mass were evaluated from four institutions.
6
ROC analysis confirmed the overall superiority of the ANNderived composite index over Ca 125 alone (p = 0.0333). At a fixed
specificity of 98%, the sensitivities for ANN and Ca 125 alone were
71% (37/52) and 46% (24/52) (p = 0.047) respectively, for detecting early stage epithelial ovarian cancer, was 71% (30/42) and 43%
(18/42) (p = 0.040), respectively, for detecting invasive early stage
epithelial ovarian cancer.
The combined use of multiple serum tumor markers through ANN
improves both sensitivity and specificity for detection of stage I epithelial ovarian cancer. The development of technologies that measure multiple serum markers simultaneously, linked to the creation of statistical
methods that enhance sensitivity without sacrificing specificity hold great
promise. But none of the test so far is approved for clinical routine.
Genomic Approaches
Loss of Heterozygosity Analysis (LOH)
LOH analyses of solid tumors evaluate multiple regions of gene that
are frequently deleted or lost in different types of ovarian tumors.
270 F Penault-Llorca
LOH studies in borderline ovarian tumors (BOT) have shown a LOH
rate less than 25% at most of the loci when compared to invasive
epithelial cancer (IEOC). However, BOTs demonstrate specific LOH
(3p13–14.3 and Xq11.2–q12) suggesting that a large subset of BOTS
may not progress to IEOC. LOH analyses has given interesting data
for the understanding of ovarian carcinogenesis but is not useful in
the clinical setting.
Comparative Genomic Hybridization Analysis (CGH)
This method involves competitive hybridization of tumor and normal
reference DNA differentially labeled with distinct fluorescent molecules to normal human metaphase chromosome spreads. Based on the
relative intensity of the two fluorescent colors, the region of the chromosome with gain or loss in gene copy number can be identified in a
single hybridization. Differences have been shown between high and
low stages, as well as BOT and IEOC. However, CGH has no interest in the clinical setting for the moment.
Transcription Profiling (cDNA Arrays)
Transcription or gene expression profiling is a large scale approach for
analyzing gene expression data, which have been largely used to identify molecular signatures and to elucidate important aspects of epithelial ovarian cancer (EOC) in order to improve the clinical
management of this disease.
7
Despite the high degree of morphological heterogeneity, epithelial ovarian cancer gene expression profiling
reflects morphology and biological behaviour. Based on multiple
studies, gene expression profiling results can be used to stratify the
four different subtypes of EOC, namely serous papillary, mucinous,
endometrioid, and clear cell carcinoma, but some overlapping gene
expression was also noted. The prediction of the response to
platinum-based chemotherapy has been also demonstrated but is not
reproducible among different platforms.
8
Several novel candidate markers for the early detection of EOC
have been identified by gene expression profiling and examples that
Molecular and Biological Diagnosis of Early Gynecologic Cancers 271
have been validated by ELISA on the protein level in serum from
EOC patients and healthy controls. Using gene ontology, genes
involved in cell cycle regulation, in the extracellular matrix, and in
immunological responses have been identified. They are relevant to
epithelial ovarian cancer biology and particular genes potentially can
serve as therapeutic targets for small molecules, biologically, and
immunologically. Gene ontology studies have permitted the creation
of gene signatures. Therefore, a limitation of DNA arrays is that transcriptional difference in the tumor does not completely reflect the
protein observed peripherally, since many protein–protein interactions
and post-translational modifications may change the protein patterns
found in the blood.
This area is still entirely in the domain of research. In fact, cross
validation among different platforms and prospective validation in
multi-center trials are necessary before microarray technology can
move to clinical practice. The potential implications of the results for
the clinical management of EOC are enormous.
Proteomics
Proteomics is the study of information flow within the cell and the
organism through protein pathways and networks of cellular protein
interactions of normal and disease state. Proteomic approaches have
been used to define a distinctive pattern of peaks on mass spectroscopy or to identify a limited number of critical markers that can
be assayed by more conventional.
Methods
Mass spectrometry allows a high-throughput study such as proteomic analysis of serum or microdissected cells. Matrix assisted laser
desorption and ionization (MALDI) with time-of-flight (TOF) detection (MALDI-TOF) and surface-enhanced laser desorption and ionization (SELDI-TOF) are two methods of mass spectrometry
currently used in proteomics. In the MALDI technique, the protein
samples of interest are immobilized in an energy-absorbing chemical
matrix on a chip or a plate and the entire proteome within the range
272 F Penault-Llorca
detectable by mass spectrometry undergoes analysis. SELDI is a
refinement of MALDI. A selective surface is used to bind a subset of
those proteins based on different properties. Both SELDI and
MALDI use laser energy to ionize and launch bound peptides (desorption) across a vacuum tube to detector plate. Time of flight is
dependent on the mass-to-charge ratio of the peptide (m/z) and the
data are recorded as m/z peaks of relative intensities.
As the profile generated by SELDI has generally up to a million
data points, bioinformatics algorithms have been developed to recognize important protein patterns.
10,11
Recently, Lopez et al.12have
identified 162 proteins from peptides and protein fragments bound
to carrier proteins from the serum samples of ovarian cancer
patient. Within this study, three sets of the discriminating carrierprotein bound fragments differentiated samples from patients with
ovarian cancer (453) and apparently healthy controls (110) with
sensitivities and specificities of up to 93% and 97%, respectively.
The proteins are involved in cellular inflammation, differentiation,
signaling, apoptosis, transcriptional regulation, and other regulatory mechanisms.
The power of this approach is 4-fold: (a) it is unbiased and
does not presuppose any particular disease mechanism, (b) multiple
differences — multiple putative disease markers, are often discovered
and combinations of markers are likely to be more powerful discriminators than single markers, (c) large numbers of samples (usually
blood sera) from appropriate cohorts can be analyzed quickly for discovery and subsequent validation of putative marker sets, and (d) the
method does not require a priori antibody development for success.
Mass spectrometry has, however, generated controversy centered
primarily on two issues: the relative importance of obtaining definitive
sequence identification for differentiating masses and the likelihood
that peptides or protein fragments, as opposed to intact proteins, can
be useful as disease biomarkers.
13
The clinical relevance of the serum
peptidome has been vigorously debated. The protein signal peaks
used to distinguish between normal and disease have not all been
identified. But recent publications have confirmed that specific protein fragments are correlated with disease stages.
14
Molecular and Biological Diagnosis of Early Gynecologic Cancers 273
A major area of controversy has been the lack of data consistency
and reproducibility across the various published studies, although
with proper attention to stringent experimental design and protocols,
these issues can be addressed in future studies.
15
Conclusions
Candidate biomarkers for early ovarian cancer have been discovered
through empirical development of monoclonal antibodies, studies of
gene expression, cloning of gene families and proteomic techniques.
Given the heterogeneity of ovarian cancer, it is unlikely that any single marker will be sufficiently sensitive to provide an effective initial
screen. Sensitivity of serum assays might be enhanced by utilizing a
panel of biomarkers. With the exception of Ca 125, all the assays
developed in this chapter need different steps of validation. In the
future, the development of clinical trials in proteomics will allow personalized medicine for IEOC. Regular serum screening tests will
diagnose cancer long before it is evident clinically, perhaps even
before it can be detected radiographically.
CERVICAL CANCER
Human papillomavirus (HPV) is the major cause of cervical cancer, and
in the natural history of the disease, persistent HPV infection precedes
the appearance of cytological abnormality. Episomal state: briefly, after
infecting the host, the virus may exist as an inactive extrachromosomal
particle (episome) that is detectable only by DNA testing (latency).
This type of infection (subclinical, DNA-only) is extremely common,
occurring in up to 50% of sexually active young women. It generally
occurs in cells where there is no cytological evidence of disease (normal
cytology); 99% of sub-clinical infections are cleared by the body’s natural immune process. The virus can become active, viral DNA is transcribed, and viral particles are assembled. The virus begins to replicate
independent in the host cell (active infection). This type of infection
results in a clinically detectable lesions including abnormal Pap smear,
genital wart. Immune response begins on average within three months
274 F Penault-Llorca
of productive viral infection. In the rare occurrence that immune
response is suppressed, progression to high-grade disease may occur.
When integration occurs, the viral DNA transforms the host
DNA. Cofactors, high-risk HPV types, and co-mutagens appear to
be necessary for this to occur. The circular HPV episome must break
into a linear strand prior to integration into host DNA. This break
often occurs at a region of HPV DNA that produces a regulatory
product: E2. The E2 product normally serves to control cell-proliferation-inducing genes E6 and E7. In the absence of E2, cell growth is
out of control resulting in neoplasm. Only 10%–20% of HPV-infected
cases are at risk for progression to neoplasia. These patients will have
either a persistent HPV infection or they will have a recurrent infection after a lesion free interval (see Chapters 2, 3 and 7).
New Markers in Cervical Cancer Screening
The study of cervical carcinogenesis has yielded a series of biomarkers
that potentially could be used as surrogates for the identification of
HPV-related disease of epithelial cells.
These biomarkers are promising for the surveillance of patients
with confirmed dysplasia and the need for either continued surveillance after preventive or therapeutic management, or surgery.
16
HPV Testing
Available assays — molecular methods
Hybrid Capture
HPV testing by Digene Hybrid Capture II (HCII) — The HCII HPV
DNA test (Digene Corporation, USA), is the only Food and Drug
Administration (FDA) approved test for detecting 13 high-risk HPV.
It is widely used to triage patients diagnosed with ASC-US (atypical
squamous cells of undetermined significance). The high sensitivity of
the HCII assay allows it to be used to triage patients with ASC-US and
a negative HCII result into routine yearly screening. However, one
Molecular and Biological Diagnosis of Early Gynecologic Cancers 275
limitation of HCII is that it can be performed only on liquid-based
cervical cytology samples. Other limitations of this test are that
(i) it does not preserve cellular morphology, (ii) analytical sensitivity
is 10,000 viral copies/mL (1.0 pg/mL), (iii) cross-reactivity between
low and high risk types = 11.7%–22%, (iv) “Blind” Test — result measured by a luminometer. However, there is no ability to confirm whether
patient or partner DNA, no ability to confirm that the HPV is responsible for cellular change and, no internal negative patient control.
Tissue Based Assays: In situ Hybridization Kits
The commercially available methods for detecting high-risk HPV in
paraffin-embedded tissue consist of in situ hybridization (ISH). Two
ISH kits are developed for research use: the DakoCytomation ISH
high-risk probe (Glostrup, Denmark) (the probe consisted of a cocktail directed against high-risk HPV types 16, 18, 31, 33, 35, 39, 45,
52, 56, 58, 59, and 68), and the Ventana INFORM HPV ISH assay
(Tucson, Arizona) which uses a nonamplified high-risk HPV probe
consisting of a cocktail directed against six HPV types 16, 18, 31, 33,
35, 51 (cross reacting with other high risk HPV 39, 45, 52, 56, 58,
and 66, but not with low risk HPV, and a low risk HPV ISH assay
(HPV types 6 and 11). ISH assays allow the detection of HPV DNA
in tissue and liquid-based cytology samples. Sensitivity is 10–50 copies
of target DNA per nucleus. The type of signal (confluent, punctate)
may reflect either episomal or integrated form of viral target DNA.
The main advantage is the ability to correlate DNA probe results with
morphology. The clinical utility is hampered by the manual, laborintensive nature of the procedure, but the assays have been recently
automated by Ventana Medical Systems, Inc.
Surrogate Markers
Proliferation Biomarkers Such as INK4A or p16
17
The E6 and E7 oncoproteins from high-risk HPV types cause chromosomal abnormalities and genomic instability. In addition, the E6
276 F Penault-Llorca
gene product mediates degeneration of the p53 tumor suppressor
protein, and the E7 gene product binds to and inactivates the
retinoblastoma protein, which results in increased production of the
CDNK2A gene product, p16
Ink4a
. p16
Ink4a
, a tumor suppressor protein that inhibits cyclin dependent kinases involved in cell cycle control, has been shown to be overexpressed in high-grade dysplasias and
cervical carcinomas. In recent years, p16
Ink4a
has been extensively
investigated as a diagnostic aid in various scenarios in gynecologic
pathology. Like with all markers, in each of these scenarios, p16
Ink4a
is neither 100% specific nor sensitive for a given lesion.
Positive p16
Ink4a
staining can be useful to differentiate between
atypical immature squamous metaplasia without CIN and some cases
of immature metaplastic squamous epithelium with associated CIN.
Low-grade CIN exhibiting p16
Ink4a
positivity is more likely to
progress to high-grade CIN than are p16
Ink4a
negative lesions. It is
possible that p16
Ink4a
could be used to triage cases of low-grade CIN,
which are associated with high-risk HPV and more careful follow up
is required. p16
Ink4a
is expressed in a high percentage of high-grade
CIN and expression within the upper two-thirds of the squamous
epithelium is a significant indicator of the presence of a high-grade
dysplastic lesion. There is poor interobserver reproducibility in the
histologic diagnosis of CIN; p16
Ink4a
immunostaining has been shown
to reduce interobserver variability in cervical biopsy specimen interpretation, especially aiding in the identification of small focal areas of
high-grade CIN. p16
Ink4a
is also a value in distinguishing high-grade
CIN from mimics such as atrophic squamous epithelium, immature
squamous metaplasia, and transitional metaplasia. p16
Ink4a
may be
combined with the proliferation marker MIB1/Ki67. Not surprisingly, most cervical squamous carcinomas also stained with p16,
because they contain high-risk HPV. Normal endocervical glands are
usually p16
Ink4a
negative with an occasional case exhibiting focal weak
positivity. In contrast, most preneoplastic and neoplastic endocervical
glandular lesions, as a result of their association with high-risk HPV,
exhibit diffuse p16
Ink4a
positivity. However, a small number of cervical
adenocarcinomas are p16
Ink4a
negative because they are not associated
with high-risk HPV, the relationship between these HPV types and
Molecular and Biological Diagnosis of Early Gynecologic Cancers 277