Diagnostic Modalities

David Starks, Bin Yang, and Peter G. Rose

In the field of gynecologic oncology, the various diagnostic modalities available serve as invaluable tools in the diagnosis, management, staging, treatment, and monitoring of gynecologic malignancies. Technological advances in existing modalities such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) have furthered their utility as diagnostic and management instruments, while the indications for newer imaging modalities such as 2-(18F)-fluoro-2-deoxy-D glucose (FDG) positron emission tomography (PET)/CT continue to expand. The use of tumor markers in identifying disease and molecular pathology in confirming which specific disease exists is presented. Because of the important role played by these diagnostic tools, the gynecologic oncologist must possess at least a passing familiarity with the basic science underlying these diagnostic instruments, as well as understand their advantages and limitations in imaging the spectrum of gynecologic cancers. Not all diagnostic modalities are useful or appropriate in evaluating the different and varied gynecologic cancers. Furthermore, the impact of diagnostic studies in the field of gynecologic oncology is ever expanding as newer technological developments become available to the clinician who must understand how to translate these advancements into improved patient care. Finally, as cost-effectiveness becomes an ever more important driver of health care decision making, it behooves the gynecologic oncologist to understand the various diagnostic tools in his armamentarium in order to use them to maximal effect.

IMAGING MODALITIES

Diagnostic imaging is an expanding field that has replaced radiology and now encompasses numerous new and varied technologies.

Ultrasound

Ultrasound is the most widely used imaging modality in the field of gynecology and is often the initial radiologic study used in the evaluation of pelvic abnormalities. Ultrasound technology uses a hand-held transducer containing piezoelectric crystals capable of emitting high-frequency sound waves that are projected into the patient’s body. Emitted frequencies range from 7.0 to 8.0 MHz, used in transabdominal scanning, and up to 9.0 MHz is generally used in transvaginal ultrasound (TVUS). Higher-frequency sound waves result in improved image resolution, but reduced tissue penetration. The piezoelectric crystals serve as both the emitter and receiver of the sound waves. As the wave encounters tissue surfaces, it is both reflected and transmitted. The reflected wave returns to the transducer, where it is converted into an electrical signal, which is termed an echo, and the signal is amplified and converted into different shades of gray based on the degree of amplification. Stronger echoes are perceived as whiter shades, whereas weaker echoes are assigned darker shades.

Doppler ultrasonography can be added to basic ultrasound studies in order to evaluate vascular structures and blood flow. Doppler ultrasonography uses the principles of the Doppler Effect, which states that a moving object will emit a wavelength with differing frequencies and lengths based on whether the object is moving toward or away from the source emitting the sound wave. The sound waves emitted by the ultrasound transducer are reflected by vascular structures being studied, and objects moving toward the transducer emit a high-frequency, short-wavelength echo, whereas objects moving away from the transducer emit a low-frequency, long-wavelength echo. Based on these frequencies and wavelengths, the ultrasound transducer is able to determine the velocity of flow in the vascular structure and generate a Doppler waveform. Color-flow Doppler, by convention, assigns flow toward the transducer as red and flow away from the transducer as blue, but this assignment is arbitrary and can be reversed by the ultrasonographer.

The strengths of ultrasound technology includes its relative ubiquity and low cost, as well as a high safety profile due to the absence of ionizing radiation. The weaknesses of ultrasound include its reliance on the skill and experience of the operator, the need for a high degree of training in order to obtain a necessary degree of competence, the inability of the sound waves to penetrate gas or bone, and difficulty in visualizing midline organs due to the obscuring effect of overlying bowel gas or patient obesity and body habitus.

Indications for performing ultrasound studies in the field of gynecologic oncology include the initial evaluation of the endometrial lining in the setting of postmenopausal bleeding; evaluating and describing the nature of pelvic and adnexal masses, identifying and diagnosing gestational trophoblastic disease, and playing an important role in the performance of ultrasound-guided biopsies and percutaneous drainage procedures conducted by interventional radiologists.

Computed Tomography

High-resolution CT scans are an invaluable tool for the gynecologic oncologist, and use of CT scans in the field is extensive. Indeed, CT scans are probably the most frequently used imaging technique ordered by gynecologic oncologists and the second most common imaging study after ultrasound in the field of gynecology. CT scans offer the oncologist a noninvasive means of evaluating the extent and spread of metastatic disease and provide guidance in surgical planning, detecting disease recurrence and progression, and detecting lymph node involvement. CT scans also play a role in interventional radiology, permitting accurate biopsies and drainage procedures to occur.

CT scanners use x-ray beams that are rotated about a patient in a 180-degree arc. Laying directly opposite of the beam emitters are sets of crystal detectors, 2 to 10 mm in size, that capture the emitted photons and measure tissue absorption. This information is then processed by a computer that generates a 2-dimensional cross-sectional representation of the anatomic structure under radiologic evaluation. In order to generate clearer and more diagnostic images, contrast medium is often used to enhance the discrepancy in tissue absorption and densities. Contrast medium may be given to patients either orally, intravenously, or rectally, and care must be used when administering contrast to patients with a known iodine allergy or renal dysfunction.

The advantages of CT scans include a short period of time required for scanning; minimization of the dependency on operator skill, thus leading to a high degree of reproducibility, and a high degree of spatial and anatomic resolution. More recent modifications in CT technology have led to further improvement in image resolution and visualization. For example, helical CT combines continuous patient transport through a scanner with a single row detector array that generates a spiraling projection of x-rays. This is a dynamic modification of the traditional CT, in which the patient remains stationary while being scanned. Helical CT decreases the scanning time required for a patient, decreases the volume of contrast dye that needs to be administered for improved resolution, and provides more images from several different angles, thus generating higher image quality. Helical CT also has improved detection of smaller lesions that are difficult to detect with conventional CT. Movement artifacts such as peristalsis in the intestines or interference from patient breathing are minimized, allowing for improved image resolution. Another modification of conventional CT is the multidetector CT scanner (MDCT), which uses the same basic principles of helical scanning, but uses a multiple-row detector array instead of a single-row detector array. Current scanners use 16-, 32-, or 64-slice systems that create a cross-sectional slice thickness of 1 to 2 mm, yielding images with even higher spatial resolution and improved image quality.

The disadvantages of CT scans include the use of ionizing radiation, which has led to a growing concern of an increased risk of radiation-associated cancers. A recent study concluded that for a 40-year-old female patient being evaluated by a routine CT of the abdomen and pelvis with contrast, the chance of developing cancer is 1 in 870, as compared with 1 in 470 for a woman 20 years of age and 1 in 1400 for a woman 60 years of age.¹ Caution must be used in interpreting these findings and those from similar studies. The risk of cancer to the individual patient from having a CT scan performed is small, even with the use of high-dose radiation. This risk is often outweighed by the benefits accrued from CT scans that are truly indicated, but the growing awareness in the medical community of this risk of cancer secondary to radiation has led to an effort to minimize nonindicated or multiple scans. Other disadvantages of CT include the fact that patient body habitus, as well as implanted metallic devices and prostheses, can obscure and degrade image quality, leading to the generation of a nondiagnostic scan.

Magnetic Resonance Imaging

MRI scanners produce a magnetic field that aligns hydrogen nuclei within the patient. An intermittent radiofrequency pulse is emitted from the scanner, which alters the alignment of these hydrogen nuclei. When the radiofrequency pulse is discontinued, the hydrogen nuclei return to their original alignment, releasing a quantity of energy in the process. The amount and rate of energy released is wholly dependent on the property of the tissues containing the hydrogen nuclei. The longitudinal relaxation time is termed T1, and in T1-weighted images, fluid appears dark and fat appears white. In the transverse relaxation time, termed T2, fluid has a white appearance. In contrast to CT, MRI does not use ionizing radiation, but instead relies on magnetic fields and radio-frequencies. The paramagnetic element gadolinium is used as a contrast agent in MRI, and in comparison with the iodine-containing contrast agents used in CT, gadolinium has a much lower rate of adverse reactions and allergies. Recent studies of gadolinium have established an association with nephrogenic systemic fibrosis (NSF) in patients with a history of acute renal failure and end-stage renal disease. The American College of Radiology (ACR) has published guidelines for the use of gadolinium in patients at risk for the development of NSF.² The ACR recommends that before the administration of gadolinium, a recent glomerular filtration rate (GFR) (in the last 6 weeks) be obtained for any patient with a history of renal disease, age greater than 60 years, history of hypertension or diabetes, or history of severe hepatic disease or liver transplantation. Although patients with stage I or stage II chronic renal disease do not require any special consideration, patients with stage III or V disease (GFR < 30 mL/min/1.73 m²) need to be referred to a nephrologist for evaluation before gadolinium administration and potential dialysis after the performance of an MRI.

The advantages of MRI include the use of nonionizing radiation and non–iodine-containing contrast agents. MRI can penetrate calcified material such as bone without significant attenuation in the signal or loss of image resolution. MRI also has remarkable soft tissue resolution. Recent advances have shortened the imaging time required to obtain scans, as well as created techniques for imaging the heart and blood vessels without the need for contrast agents.

The disadvantages of MRI include the expense of scans, issues with motion artifacts, and the inability of patients with metallic implants and prosthesis, such as pacemakers and artificial joints, to be placed inside an MRI scanner. Many patients experience episodes of anxiety or claustrophobia inside MRI scanners and may require anxiolytics to be fully compliant during scanning.

The role of MRI in gynecologic oncology is still being elaborated, but it can serve as a useful adjunct in determining the extent of local and distant tumor spread in gynecologic malignancies and also has great sensitivity and specificity in the evaluation of vaginal and vulvar cancers.

Positron Emission Tomography

PET scans have developed an important role in the diagnosis and management of a number of different malignancies, including gynecologic cancers. PET scans use a radiochemical tracer, most commonly FDG, which is preferentially taken up by malignant cells due to their increased rate of glycolysis. This means that PET scans are capable of detecting the early biochemical abnormalities associated with malignancy, before the development of the structural and tissue changes caused by malignancies that are necessary for cancer detection by other imaging techniques.³ Combining FDG-PET with CT in a single scanning device has allowed the images obtained by both modalities to be fused, generating images in which areas of increased FDG uptake are superimposed on CT images, providing anatomical localization.

The advantages of PET scans are largely due to their ability to detect the early abnormalities associated with tumor growth and recurrence. Their disadvantages include the current high cost of both the PET scanner, as well as the cost of the scan. These costs are often not covered by a patient’s insurance, except in a few indicated conditions such as staging cervical cancer. PET scans have a high false-negative rate in evaluating lesions that are less than 1.0 cm in size or in detecting malignancies with low metabolic activity. Furthermore, areas of inflammation can result in false-positive results.

The role of FDG-PET CT in the management of gynecologic cancers is still being elaborated. It currently has a role in the staging of cervical cancer, as well as monitoring for recurrence of cervical cancer. PET may also be useful in the detection of recurrent ovarian and endometrial cancer, but more study is needed before greater clinical application is undertaken.

Image-Guided Percutaneous Biopsies

The use of percutaneous biopsy, performed by interventional radiology using either CT or ultrasound guidance, to assist in the diagnosis of a pelvic mass continues to develop and is not without controversy. Biopsies may be obtained with the use of 16- or 18-gauge needles or through the use of 1.0- to 1.8-mm Surecut needles (UK Biopsy Ltd., Halifax, Great Britain) in order to obtain a core biopsy. Clinicians have raised concerns that biopsies of cystic ovarian lesions may have a high false-negative rate and a low diagnostic accuracy, while increasing a patient’s risk for procedure-related tumor seeding and contamination of the peritoneum due to rupture or leakage of the cystic mass. It may be that the concern for peritoneal seeding of malignancy is theoretical at best, but the current clinical consensus in the management of a cystic ovarian lesion is for surgical management of the lesion, either by laparoscopy or laparotomy, rather than through percutaneous biopsies. The use of percutaneous biopsies to aid in the diagnosis of solid pelvic masses is less controversial and may be of benefit, especially in cases of widespread meta-static disease. The concern for intraperitoneal seeding does not appear to have the same risk for tumor leakage that seems inherent in performing a biopsy in a cystic lesion. Ascites is often a common finding in the setting of advanced malignancy, but the sensitivity of performing cytology on ascites appears to be 60%. With solid tumors, it is possible to obtain larger tissue samples by core biopsy, permitting immunohistochemical studies and molecular profiling to be performed. A recent study by Hewitt et al⁴ examined 149 women with suspected ovarian cancer undergoing a biopsy of an adnexal mass by either CT or ultrasound guidance using an 18-gauge needle. The diagnostic rate was 90% for CT and 91% by ultrasound, with only 1 hemorrhagic complication documented in the series. The authors argued that percutaneous biopsy was safe and could be considered as a replacement for surgical intervention in the initial management and diagnosis of malignancy. Such conclusions are currently preliminary at best, and surgical management and diagnosis of pelvic masses is still considered to be the standard of care.

IMMUNOHISTOPATHOLOGY

Immunohistochemistry is a method for localizing specific antigens in tissue or cells based on antigen-antibody recognition. In the past 3 decades, a laundry list of antibodies has been developed with tissue specificity. Immunohistochemistry has enormous impact on the accuracy of pathologic diagnosis. We briefly summarize the current application of immunohistochemistry in facilitating the accurate diagnosis of gynecologic neoplasms, with focus on malignant epithelial neoplasms.

Cervix: p16 Is a Surrogate Biomarker for High-Grade Cervical Dysplasia

High-risk human papilloma virus (HPV), most commonly 16 or 18, is responsible for the majority of cervical cancers. Development of invasive cervical cancer is preceded by HPV-related cervical dysplasia, known as cervical intraepithelial neoplasia (CIN). CIN can be low grade (CIN1) or high grade (CIN2-3). Detection of high-grade cervical dysplasia by Pap smear and subsequent tissue biopsy is critical in the identification and treatment of precursor lesions for the prevention of cervical cancer. Unfortunately, the reproducibility of diagnosis of CIN2 in pathologic samples is not good. Therefore, the identification of surrogate markers for HPV infection and, more importantly, evidence of molecular changes leading to cervical cancer would be of vital importance in the discrimination between low-grade and high-grade dysplasia.

These HPV types encode 2 proteins, E6 and E7, that are oncogenic by their inhibition of tumor suppressor genes. This results in uninterrupted cellular replication and malignant transformation of some infected cervical cells.⁵ Integration of HPV DNA into the cells is the prerequisite step for the expression of the oncoproteins E6 and E7, which subsequently degrades tumor suppressor proteins such as p53 and pRb. HPV E7 protein expression results in degradation of pRb protein, which normally inhibits the transcription of p16 (CDKN2A).

Diffuse p16 expression has reliably been shown to be a surrogate biomarker for CIN2-3. However, there are approximately less than 30% of CIN1 lesions also expressing p16 with a expressing p16 with a focal and patchy pattern. Recent studies indicated that approximately 20% of p16-positive CIN1 progress to high-grade lesions as compared with none of the p16-negative CIN1 lesions within a 12-month follow-up period. Diffuse and full thickness of the p16 immunostaining pattern is a hallmark of high-grade CIN and is a very useful ancillary tool in those challenging cases in differentiating CIN2 from CIN1 and from immature squamous metaplasia.

Diffuse expression of p16 is also seen in adeno-carcinoma in situ (AIS) of the cervix. Again, this expression correlates with the involvement of high-risk HPV in the development of these lesions. Detection of p16 is helpful in the diagnosis of cervical AIS to distinguish AIS from endometriosis and tuboendometrial metaplasia. The latter has a focal and discontinuous staining pattern that is distinct from the disuse and continuous staining pattern in AIS lesions. Additionally, p16 aids in the differential diagnosis of endometrioid adenocarcinoma of the endometrium with endocervical adenocarcinoma. A diffuse p16 staining pattern is typically seen in endocervical adenocarcinoma, but is rarely seen in endometrioid adenocarcinoma. However, it should be emphasized that p16 immunostaining alone has no role in the differential diagnosis between endocervical adeno-carcinoma and serous adenocarcinoma of the endometrium because diffuse p16 staining pattern can be seen in both types of cancer.

Vulva: p16 and p53 Expression in Vulvar Intraepithelial Neoplasia

There are 2 types of vulvar intraepithelial neoplasia (VIN), classic (usual) and simplex (differentiated) types, based on histopathologic features and distinct molecular pathogenetic pathways. The most common precursor for vulvar squamous cell cancers is the classic or usual type of VIN, which is associated with HPV infection. These tumors are seen in younger patients who often have a history of cervical HPV infection. It has shown that tumor suppressor protein p16 has been overexpressed in the majority of high-grade VINs. The staining pattern is diffuse and full thickness of the dysplastic epithelium. The molecular mechanism for overexpression of p16 in the classic type of VIN is analogous to that seen in HPV-associated cervical CIN.⁶ Furthermore, unlike simplex VIN, classical VIN is rarely associated with p53 overexpression.

Simplex (differentiated) type of VIN is less frequently seen clinically and tends to be seen in older patients with no association of HPV infection. These lesions often arise in a background of lichen sclerosis. Histopathologically, recognition of simplex VIN and differentiating it from benign squamous hyperplasia can be challenging. Furthermore, these lesions are not as commonly detected before the development of invasive disease. This is thought to reflect both the difficulty in clinical and pathologic diagnosis of this lesion and the fact that it is thought to have a short time to progression to invasive disease.

Simplex VIN and keratinizing squamous cell vulvar cancers have consistently been strongly associated with p53 mutations. It has been shown that approximately two-thirds of simplex VIN lesions display overexpression of p53 immunohistochemically. p53 immunostaining is a useful ancillary tool in making the distinction between simplex VIN and benign vulvar lesions. However, caution must be taken when dealing with a lesion with morphology and a p53 immunostaining discrepancy. Because p53 deletion is seen in some of the simplex VINs, a negative p53 immunostaining should not prevent the diagnosis if morphologically convincing.

Immunoprofile of Extramammary Paget Disease

Extramammary Paget disease (EMPD) is an unusual diagnosis characterized by the presence of Paget cells proliferating within the intraepidermis. Vulvar Paget disease can be primary or secondary. Primary disease is that which originates from the epidermis or skin appendages, and secondary disease is that which represents extension of a visceral carcinoma to the vulva, most commonly rectal or urologic carcinomas. The clinical and histologic appearance of primary and secondary EMPD is similar, and thus differentiation can be a challenge. The prognostic implications between the 2 diagnoses are significant. Primary EMPD is usually a locally confined lesion, and the clinical outcomes are substantially better. Secondary EMPD, on the other hand, represents the spread of visceral tumor onto the vulvar skin and has a substantially worse prognosis. Therefore, accurate clinical diagnosis is very important in this disorder. Molecular markers have recently been found to be of utility in the distinction between primary and secondary EMPD and may have utility in aiding with diagnosing these lesions.

Cytokeratins (CKs) have utility in the detection of certain cancers. CK20 is associated with colorectal adenocarcinomas. It has also been correlated with the presence of a primary colorectal adenocarcinoma associated with secondary EMPD. Furthermore, CK20 staining was not seen in primary EMPD. CK7, on the other hand, has been consistently seen in primary EMPD and sometimes in secondary EMPD, although not as frequently. Therefore, a vulvar Paget disease possessing the immunoprofile of CK7–/CK20/+should prompt an aggressive search for an underlying malignancy, such as colorectal cancer.

Gross cystic disease fluid protein (GCDFP)-15 is a glycoprotein expressed in apocrine epithelial cells. GCDFP-15 has been found in primary EMPD, and its absence is correlated with the presence of secondary EMPD.⁷ The immunohistochemical detection of carcinoembryonic antigen (CEA) has also been found to be of value in EMPD. The value of CEA staining seems to be in differentiating EMPD from superficial spreading melanoma and not in the separation of primary and secondary EMPD, although negative CEA staining is seen more commonly in secondary EMPD.

Endometrium

Based on the degree of malignancy and prognosis, endometrial carcinoma is divided into type 1 and type 2 cancers. Type 1 cancer includes endometrioid and mucinous adenocarcinoma, whereas type 2 encompasses serous and clear cell adenocarcinoma.

Loss of PTEN in Type 1 Endometrial Carcinoma

In type 1 cancers, loss of expression of pTEN protein due to point mutations and promoter methylation is the most frequent genetic alterations observed. PTEN is a tumor suppressor gene located on chromosome 10q23. The PTEN gene encodes a dual-specificity phosphatase with a role in cell cycle arrest and promotion of apoptosis via phosphatidylinositol-(3,4,5)-triphosphate (PIP3). Immunohistochemically, the majority of endometrioid and mucinous adenocarcinomas have negative immunoreactivity to pTEN antibody compared with adjacent benign endometrium. Because loss of pTEN is an early molecular event during endometrial carcinogenesis, lack of pTEN immunoreactivity has in recent years become the important biomarker in identifying the precursor lesion of endometrial intraepithelial neoplasia and a small subset of higher-grade endometrioid adenocarcinomas acquiring p53 mutations at a late stage.⁸ Therefore, p53 immunostaining alone does not distinguish between serous adenocarcinoma and grade 3 endometrioid adenocarcinoma harboring a p53 alteration.

p53, p16, and IMP3 Expression in Type 2 Endometrial Carcinoma

Type 2 cancers are often characterized by p53 mutations. p53 is a tumor suppressor gene and is the most commonly mutated gene in human cancers. p53 protein product binds to DNA and upregulates transcription of genes, which act to halt the cell cycle and assist with DNA repair or initiate apoptosis if repair is not possible. Anti-p53 antibody reacts with both wild-type and mutant p53 proteins. However, because the half-life of wild-type p53 protein in cells is only less than 20 minutes, it rarely detects p53 immunoreactivity in most normal cells. In contrast, because the majority of mutant p53 proteins have greater than 16 hours of half-life, its accumulation can be easily seen immunohistochemically in malignant cells. Approximately 90% of serous adenocarcinomas demonstrate p53 mutation and accumulation of p53 immunohistochemically. Different from type 1 cancers, p53 mutation is an early genetic event in serous adenocarcinoma. It has been shown that up to 80% of endometrial intraepithelial carcinomas (EIC) contain mutations in p53. Therefore, immunohistochemical detection of p53 overexpression is a very useful biomarker in identifying and confirming early precursors of EIC in endometrial biopsy or curettage specimens. Approximately 30% to 40% of clear cell adenocarcinomas harbor p53 mutations.

Overexpression of p16 protein is also seen in serous adenocarcinoma of the endometrium or the ovary. Overexpression of p16 in serous adenocarcinoma is not linked to HPV infection. The underlying molecular mechanism is still largely unresolved. Because both cervical adenocarcinoma and serous adenocarcinoma can share high nuclear grade histopathologically, and overexpression of p16 is found in both cervical adenocarcinoma and serous adenocarcinoma, another layer of challenge in the differential diagnosis of the 2 is added. When the issue arises, application of both p16 and p53 immunostains helps in resolving the issue. Serous adenocarcinoma will be strongly positive both for p53 and p16, whereas cervical adenocarcinoma will be positive for p16 but negative for p53.

IMP3 is an oncoprotein that is mainly expressed in fetal and malignant tissues and rarely in adult benign tissues. IMP3 has been found to be involved in cell growth, adhesion, and migration. Strong IMP3 staining has been demonstrated in 86% to 94% of serous adenocarcinoma, as opposed to only 3% to 28% of endometrioid adenocarcinoma. Furthermore, 50% of clear cell cancers also stain positive for IMP3. Expression of IMP3 is also seen in 89% of EICs, indicating its involvement in early carcinogenesis.

Ovarian Epithelial Cancers

There are 5 types of ovarian epithelial cancer, each with distinct cell types and clinicopathologic features and treatment options: (1) papillary serous carcinoma (PSC), (2) clear cell carcinoma, (3) endometrioid carcinoma, (4) mucinous carcinoma, and (5) transitional cell carcinoma. Each type has its distinct pathogenesis and immunophenotypes.

p53 and p16 Expression in Papillary Serous Carcinoma

p53 mutations are seen in approximately 70% of high-grade PSCs, but are rarely seen in low-grade PSCs.⁹ p16 is also expressed in high-grade PSC. The similar expression pattern of p53 and p16 in both endometrial and ovarian serous adenocarcinoma suggests an analogous pathway for carcinogenesis of these tumors.

Immunoprofile for Mucinous Adenocarcinoma

Mucinous adenocarcinoma, especially intestinal type, of the ovary can be morphologically indistinguishable from those derived from gastrointestinal tract. One of the most important issues clinically is to know whether a mucinous adenocarcinoma is primary ovarian cancer or secondary from other sites. Earlier studies indicate that the majority of colorectal cancer is CK7 negative and CK20 positive, whereas primary ovarian mucinous carcinoma is positive for both CK7 and CK20. Therefore, a CK7-negative mucinous adenocarcinoma is likely metastasized from the colorectum, and a CK-positive mucinous adenocarcinoma is likely an ovarian primary, if endocervical adenocarcinoma is excluded. The recent discovery of CDX2 expression in most of gastrointestinal cancers further facilitates the differential diagnoses. However, recent evidence of expression of CDX2 in some of the primary mucinous adenocarcinoma of the ovary and the expression of CK7 in some right-sided colon cancers further complicated the case. Therefore, although immunohistochemistry is in many situations helpful in the differential diagnosis, it is by no means a magic bullet. Clinicopathologic correlation is still crucial in rendering the correct diagnosis. Furthermore, CK7-negative and CK20-positive immunoprofile is also seen in mucinous adenocarcinomas of the lung, breast, pancreas, and stomach. Therefore, immunohistochemical findings must be put into a clinical context, and the expression of CK7 does not exclude that the ovarian tumor is secondary.

TUMOR MARKERS

Tumor markers are serologic substances that are produced by a malignancy or are abnormally elevated in response to the presence of a malignancy. They can be enzymes, growth factors, hormones, tumor antigens, receptors, and glycoconjugates. They play a role in screening, diagnosis, monitoring treatment response, and detecting disease recurrence. A large number of tumor markers have been investigated in recent years, but few have entered into clinical practice. This has largely been due to the fact that many of these tumor makers have poor specificity, which is defined as the proportion of patients without a cancer who have a negative test. Many of the current markers can be elevated in a number of conditions, both benign and malignant, contributing to their lack of specificity. With the development of new high-throughput approaches such as proteomics, which uses mass spectrometry (MS) techniques, a new interest has developed in investigating the patterns of tumor marker expression. Identification and describing these patterns offers the promise of the development of more sensitive and specific assays for various cancers and their histologic subtypes.

Cervical Cancer

No serum tumor markers exist or are being researched to screen for cervical cancer due to the success of Pap and HPV DNA-based screening programs. The few markers that were historically investigated were abandoned due to their low sensitivity and specificity. Several serologic markers have been assessed to play a potential role in determining prognosis, detecting recurrent disease, and monitoring treatment response. For example, squamous cell carcinoma antigen (SCCA) has moderate sensitivity when elevated in the setting of cervical cancer, but unfortunately has a low specificity, as SCCA levels may be elevated in a number of other squamous cell cancers, such as carcinoma of the head, neck, and lung. SCCA can also be elevated in benign conditions as well, such as psoriasis and eczema. However, there does appear to be a correlation of SCCA with prognosis and the clinical response of cervical cancer to treatment. Levels of SCCA above 1.1 ng/mL are associated with a poor prognosis. A recent study demonstrated that elevated levels of SCCA immediately after treatment with chemoradiation was predictive of distant recurrence.¹⁰ Another study demonstrated that in patients being treated with chemoradiation, previously elevated SCCA levels normalized in 93% of patients at 1 month after treatment and in 96% of patients with a complete remission at 1 month. Although used in Europe and Japan, no US company has pursued licensing of this assay for use in the United States.

CA-125 is elevated in only approximately 21% of women with squamous cell carcinoma of the cervix and may correspond to prognosis, particularly if there is a decrease in preoperative CA-125 after treatment. The addition of other markers to CA-125, such as CEA and CA19-9, can increase the sensitivity of a tumor marker panel for detecting cervical cancer, but currently, obtaining these 3 tumor markers to manage a diagnosed cervical cancer is not considered to be standard of care.

CA-125 is elevated in 20% to 75% of patients with cervical adenocarcinoma and may reflect tumor stage, size, grade, presence of lymphovascular space involvement and lymph node involvement. A recent study of patients with adenocarcinoma of the cervix observed that in multivariate analysis, CA-125 was an independent prognostic factor for disease-free survival. The investigators also demonstrated that tumor necrosis factor receptor type I may potentially be the most useful marker in evaluating the prognosis of adenocarcinoma, particularly in early-stage disease.¹¹ Further research into the use of these particular tumor markers is warranted before their use can become widespread.

Endometrial Cancer

CA-125 is often elevated in patients with uterine serous carcinomas and advanced-stage endometrioid cancers, and obtaining CA-125 preoperatively has demonstrated a correlation with metastatic disease and extrauterine spread. However, CA-125 can be falsely positive, particularly after pelvic or abdominal radiation. Several other serologic markers, including CA15-3, CA19-9, CA72-4, cancer associated serum antigen, CEA, squamous-cell carcinoma antigen (SCCA), gamma-GT, urinary gonadotropin fragment (UGF), placental protein 4 and others, have been investigated as potential tumor markers but have been largely abandoned as viable candidates for either screening or clinically managing endometrial cancer. Other novel makers currently under investigation include the glycoprotein YKL-40, which, obtained preoperatively, may detect endometrial cancer and aid in determining prognosis. Higher levels of serum inhibins, particularly inhibin β-B (INH-β-B) was observed in grade 3 endometrial cancers compared with grade 2 cancers. In one study, inhibin α (INH-α) was an independent prognostic factor for progression-free survival, cause-specific survival, and overall-survival. Elevations in human kallikrein 6 may be overexpressed in patients with papillary serous endometrial cancer. Finally, pyruvate kinase M2, chaperonin 10, and α-1-antitrypsin performed well as a panel of biomarkers demonstrating a high sensitivity, specificity, and positive predictive value (PPV) in detecting endometrial cancer.¹² Testing of current biomarker candidates and the development of better tumor markers is ongoing.

Ovarian and Fallopian Tube Cancers

CA-125 is a 200-kilodalton (kDa) glycoprotein that is recognized by the OC-125 murine monoclonal antibody. It has 2 important antigenic domains: A is the domain-binding monoclonal antibody OC125; B is the domain binding monoclonal antibody M11. A number of assays exist for detecting serum CA-125; one of the most widespread is the second-generation heterologous CA-125 II assay, which uses both OC125 and M11 antibodies. The upper limit of normal of serum CA-125 of 35 U/mL was chosen because only 1% of healthy women had a value above this point. Levels can fluctuate, however, based on the phase of the menstrual cycle and is more often elevated if the woman is pre- versus postmenopausal. Eighty-five percent of women with epithelial ovarian cancer have a CA-125 level greater than 35 U/mL, with 25% to 50% of stage I patients having an elevated level and 90% of women with advanced-stage disease demonstrating an elevation in CA-125. The sensitivity of CA-125 is approximately 78%, with a specificity of 95% and a PPV of 82% based on both prospective and retrospective data. CA-125 is nonspecific and can be elevated in a number of other malignancies (cancers of the breast, colon, lung, and pancreas), benign conditions, endometriosis, pelvic inflammatory disease, and pregnancy. Finally, CA-125 is not a marker for nonepithelial ovarian malignancies, and low levels are common in borderline, endometrioid, clear cell, and mucinous epithelial tumors.

CA-125 is useful to measure treatment response, with the levels and pattern of CA-125 being monitored over time. In assessing treatment response, a decrease in CA-125 by 50% correlates with disease responsive to chemotherapy, whereas a doubling of CA-125 from baseline constitutes treatment failure and disease progression. A recent study found that CA-125 half-life and nadir concentration had independent prognostic value for disease-free and overall survival. The same investigative group recently demonstrated that a bi-exponential CA-125 decay was an indicator of poor prognosis after primary chemotherapy.¹³ However, CA-125 is a poor marker of small-volume disease and can be falsely negative, as demonstrated in studies of second-look laparotomy after chemotherapy, which found active disease despite low or normal CA-125 levels.

CA-125 is a strong predictor of disease recurrence as well, and serial measurements are used to monitor patients for this indication. Serum elevations may be detectable 2 to 6 months before evidence of disease recurrence becomes visible on imaging studies.¹⁴ A recent study demonstrated that in patients with a complete clinical remission, a progressive low-level increase in serum CA-125, from a baseline nadir, with an absolute increase of 5 to 10 U/mL, was strongly associated with disease recurrence. However, a recent randomized trial of early detection of recurrent disease with CA-125 failed to improve overall survival.¹⁵ The only other marker with current US Food and Drug Administration approval for detection of recurrent ovarian cancer is HE4. A recent study demonstrated that HE4 correlated with a patient’s clinical status in 76.2% of cases, which was not inferior to CA-125’s correlation of 78.8%.¹⁶ Use of both CA-125 and HE4 in a combined assay appears to increase sensitivity and maintain specificity in the detection of primary ovarian cancer in women with a newly diagnosed pelvic mass.

Although playing an important role in ovarian cancer surveillance strategies, the use of CA-125 as a screening tool remains investigational. Because of its lack of specificity, use of CA-125 alone as a screening tool is controversial. It has been reported that screening specificity can be increased with the addition of TVUS, improving specificity to 99.9% and PPV to 26.8% in postmenopausal women. However, the recent Prostate, Lung, Colorectal and Ovarian (PLCO) screening trial demonstrated the limitations of CA-125 and TVUS in screening for ovarian cancer. In this trial, 34,261 postmenopausal women were randomized to receive annual CA-125 and TVUS screening for 3 years, followed by 2 additional years of CA-125 monitoring. Women were referred to a gynecologic oncologist if either the CA-125 or TVUS were abnormal. The PPV for CA-125 was 3.7% and for TVS was 1%. If both were abnormal, the PPV was 23.5%, but more than 60% of ovarian malignancies would have been missed using this strategy. Finally, the sensitivity for detecting early-stage disease was notably low, with only 21% of detected ovarian cancers being either stage I or II.¹⁷

Improved screening PPV and specificity can be obtained by abandoning the use of a fixed cutoff of 35 U/mL and using the statistical Risk of Ovarian Cancer Algorithm (ROC), which uses a woman’s age-specific incidence of ovarian cancer and CA-125 behavior over time to estimate a woman’s risk of ovarian cancer. A prospective randomized trial of the ROC algorithm in 13,582 postmenopausal women demonstrated a high specificity of 99.8% and a PPV of 19% in detecting primary invasive epithelial ovarian cancer.¹⁸ The ROC algorithm is currently being used in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) study, which is ongoing at this time. In addition to CA-125, a large number of serum markers have been studied and evaluated, but very few have any relevance to the clinical realm due to poor sensitivity and specificity. CEA is elevated in endometrioid and Brenner tumors and occasionally in mucinous tumors. CA19-9 is expressed by mucinous ovarian cancers and cancers of the colon, but has low levels of expression in other epithelial ovarian cancers. A few of the tumor markers used in the diagnosis and management of nonepithelial ovarian cancers are detailed in Table 3-1.

Serum inhibin levels are elevated in sex–cord stromal tumors, especially granulosa cell tumors. Inhibin plays a role in the regulation of follicle-stimulating hormone secretion by the pituitary. It is composed of an α subunit and 1 of β subunits (BA or BB). Although inhibin A and inhibin B levels can both be elevated in patients with granulosa cell tumors, an inhibin B level is elevated in a higher proportion of these tumors. A recent study evaluating the use of serum inhibin levels in 30 women with granulosa cell tumors demonstrated that the sensitivities and specificities for inhibin A were 67% and 100% and for inhibin B were 89% and 100%, respectively. The investigators also noted that inhibin A level was elevated before or at the time of first clinical recurrence in 58% of patients, whereas inhibin B level was elevated in 85%. The lead time from elevation of inhibin levels to clinical recurrence was estimated to be 11 months. Inhibin A and B levels were not elevated in any of the 17 patients who were postoperatively disease-free.¹⁹

Vulvar and Vaginal Cancers

The rarity of vulvar and vaginal cancers has made the investigation and development of useful tumor markers considerably difficult. There are a couple of novel markers under investigation, including carbonic anhydrase IX (CAIX), which may be elevated in vulvar cancer and may be correlated with recurrence-free survival compared with CAIX-negative tumors. CAIX overexpression may also correspond to tumor progression and inguinal lymph node metastasis. Overexpression of another marker, COX-2, may correspond with disease-specific survival in vulvar cancer. Other markers that have been investigated include tissue polypeptide-specific antigen, SCCA, and urinary gonadotropin fragment. No serologic marker to date has demonstrated sufficient sensitivity or specificity to play a role in screening or in detecting recurrent disease, or in directing clinical management. Due to the rarity of these 2 cancers, the creation of large trials sufficient to validate any tumor marker under current investigation seems doubtful.

IMAGING FOR SPECIFIC GYNECOLOGIC CANCERS

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