Second-look operations are only to be conducted as part of a research study.

Among the novel biomarkers is Human Epididymal Protein 4 (HE4) which is over-expressed in ovarian cancer. HE4 is combined with CA-125 and used to monitor the progression and recurrence of disease. Similarly kallikreins, which are serine proteases have been shown to be involved in angiogenesis, tissue invasion, and cell growth. Kallikreins have also been used in combination with CA-125 (1). Other potential biomarkers include VCAM-1, Insulin-like Growth Factor II (IGF-II), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), and Epidermal Growth Factor Receptor (EGF-R). Inflammatory molecules such as cytokines such as IL-8, IL-6, and M-CSF facilitate communication between these cells of the tumor microenvironment. (3). Additional biomarkers include prolactin, osteopontin, leptin, lysophosphatidic acid, apoliprotein A1 transthyretin, COOH osteopontin fragments, and eosinophil-derived neurotoxin. Some of these factors have shown elevations in specificity and sensitivity for detection of ovarian cancer (2).

(1) Dutta S, Wang FQ, Fishman DA. The dire need to develop a clinically validated screening method for the detection of early-stage ovarian cancer. Biomark Med. 2010; 4(3): 437-439.

(2) Rein BJD, Gupta S, Dada R, Safi J, Michener C, Argawal A. (Review Article) Potential markers for detection and monitoring of ovarian cancer. Journal of oncology. Vol 2011

(3) Iijima J, Konno K, Itano N. Inflammatory Alterations of the Extracellular Matrix in the Tumor Microenvironment. Cancers. 2011; 3: 3189-3205

In addition to the neoplastic mass itself causing measurable changes, the surrounding parenchymal cells also contribute. The microenvironment constitutes interactions of tumor cells, leukocytes and stromal fibroblasts that in some instances promote tumor development and progression (1). For example matrix metalloproteinases (MMP), which are enzymes involved in tumor invasion, represent one class of factors that is produced by cells other than the tumor. Within the peritoneal cavity, initial ovarian cancer metastasis occur by detachment of cells from the ovary and then adhesion of cancer cells to the mesothelial lined peritoneum and serosa of distant organs followed by extracellular matrix degradation and then invasion into the normal host stroma (2). This stromal invasion is facilitated by MMP’s. Hundreds of other circulating factors also play a role in adhesion and invasion and as such have potential as biomarkers of ovarian cancer. A myriad of potential biomarkers have been found. Many of them have shown promising results for clinical use in early ovarian cancer detection.

(1) Barnas JL, Simpson-Abelson MR, Yokota SJ, Kelleher RJ, Bankert RB. T cells and stromal fibroblasts in human tumor microenvironments represent potential therapeutic targets. Cancer Microenviron. 2010; 3: 29-47

(2) Tan DSP, Agarwal R, Kaye SB. Mechanisms of transcoelomic metastasis in ovarian cancer. The Lancet Oncology. 2006; 7: 925-934

Preoperative evaluation of adnexal masses has been performed by several methods. Notable among these are the noninvasive diagnostic radiologic modalities such as transabdominal and transvaginal gray scale sonography, 3-dimensional sonography, color and power Doppler sonography, computed tomography (CT), magnetic resonance imaging, and positron emission tomography (PET).

Computed tomography and MRI has not been clinically useful for characterization of the adnexa. They may be used to locate large solid masses and distinguish benign from frankly malignant ovarian tumors, with overall accuracy of 88% to 93% (1). However, cross sectional imaging appear less accurate for borderline ovarian tumors and small adnexal masses. A recent study using PET/CT showed that detection of the areas of abnormal increased metabolic activity considered highly suspicious for malignant tumors in preoperative discrimination of benign versus malignant ovarian diseases with sensitivity of 100% and specificity of 92.5% (2). However, because of relative expense and limited availability as well as possible delays in referral and surgery, routine use of PET/CT is not recommended in this setting.

Transvaginal sonography (TVS) is the initial diagnostic modality of choice for the evaluation of most pelvic masses. However, the sensitivity and specificity of TVS for the definitive diagnosis of ovarian cancer are limited. Because of this, the differential diagnosis of morphologically suspicious adnexal masses, especially in postmenopausal women, typically includes ovarian cancer. Conventional sonographic criteria for diagnosing ovarian cancer, is based on the morphological classification of ovarian masses. Malignancy is unlikely in those simple cysts with smooth walls, but the presence of a solid mass or solid projections into the cyst cavity significantly increases the risk of malignancy.

Sonography often fails to differentiate between benign and malignant lesions (3). The accuracies of gray scale sonography and color Doppler imaging for distinguishing malignant from benign tumors are 80% to 83% and 35% to 88%, respectively (4, 5). As the result of these studies, many morphological sonographic scoring systems have been developed, including features such as the presence of papillary projections or irregular or thick septae (6-7). However, results of a meta-analysis provide scientific evidence that sonographic techniques that combine gray scale morphologic assessment with tumor vascularity imaging information in a diagnostic system are significantly better in ovarian lesion characterization than Doppler arterial resistance measurements, color Doppler flow imaging, or gray scale morphologic information alone (8).

Detection of early stage EOC is difficult. In multiple studies, sonography has not been proven to decrease mortality from ovarian cancer (9, 10). The reason for this is likely multifactorial, however, any means of improving visualization of the ovary, may be helpful in the detection of early lesions. The following innovative sonographic techniques are currently used to improve identification of early stage EOC.

Ultrasound Contrast Imaging - Contrast-enhanced sonography is a great tool for detection and characterization of angiogenesis. Ultrasound contrast agents for intravenous use consist of small, stabilized microbubbles usually on the order of 1-10 microns in diameter (11). These bubbles cause increased echogenicity and are thus termed, echoenhancers. The agents create this effect by causing an acoustic impedance mismatch with the adjacent tissues. This, in turn, causes increased scattering and reflection of the sound beam, thereby leading to increased sonographic signal and increased echogenicity. The degree of echo-enhancement depends on a multitude of factors including the size of the microbubble, the concentration of contrast agent, and the compressibility of the bubbles as well as the interrogating ultrasound frequency (11).

Pulse Inversion Harmonic Imaging (PIH) - In Pulse Inversion Harmonic imaging two pulses are transmitted down each ray line. The first is a normal pulse, the second is an inverted replica of the first so that wherever there was a positive pressure on the first pulse there is an equal negative pressure on the second. Any linear target such as soft tissues responds equally to positive and negative pressures will reflect back to the transducer equal but opposite echoes. Microbubbles respond in non-linear fashion and do not reflect identical inverted waveforms. This allows the separation of the fundamental component of the bubble echoes from the background, improving resolution, and increasing sensitivity to contrast agents.

Microvascular Imaging (MVI). Pulse Inversion Harmonic Imaging has led to the ability to image individual bubbles in small vessels within and adjacent to tumors with very low blood flow rates. MicroVascular Imaging allows to capture and track the bubbles as they go around and through these small leaky vessels, providing improved visualization of slowly perfused tumors. The MIP technique involves selection of maximum pixel values throughout consecutive, PIH images as the bubbles enter of replenish replenish the imaging plane. A composite image showing the vascular architecture is constructed and can be used to improve aour abitily to detect areas of abnormal vascularity.

Flash Contrast Imaging While the ability to visualize microvascular blood flow in real-time is a significant advancement, the ability to destroy contrast at will, also has diagnostic potential. By destroying the contrast within the scanplane, a “negative bolus” of contrast is created locally. Then, the time it takes for contrast to refill the scan plane and the amount of the contrast in the ROI may be used for estimation of microvessel cross-sectional area, blood flow velocity and tumor perfusion (12).

Contrast enhanced sonography may significantly improve the diagnostic ability of ultrasound to identify early microvascular changes that are known to be associated with early stage ovarian cancer (11, 13). Currently, contrast agents play a pivotal role in the imaging modalities of computed tomography (CT) and magnetic resonance imaging (MRI) by increasing image conspicuity. By increasing the density or signal intensity of a particular organ and thus, the signal to noise ratio, contrast agents help to detect and characterize parenchymal lesions. Indeed, contrast agents have received such widespread acceptance, that a CT exam performed without intravenous contrast for many indications is now considered limited. Our preclinical studies demonstrated that the intravenous contrast agents in ultrasound holds great promise in a multitude of potential clinical applications, especially in identifying aberrant vascular changes associated with malignancy (11, 14-18).

Previous studies have addressed the use of contrast-enhanced sonography for benign and malignant tumors by showing greater enhancement of malignant tumors on Doppler imaging. According to Kupesic et al the use of a contrast agent with three-dimensional power Doppler sonography showed diagnostic efficiency (95.6%) that was superior to that of nonenhanced three-dimensional power Doppler sonography (86.7%) (19). However, simple documentation of tumor enhancement may not be sufficient because some benign tumors show detectable contrast enhancement. This limitation can be addressed by assessment of the contrast enhancement kinetics. Only 2 studies have been published that used kinetic parameters of the contrast agent to compare benign with malignant tumors in the power Doppler mode. Ordén et al demonstrated that after microbubble contrast agent injection, malignant and benign adnexal lesions behave differently in degree, onset, and duration of Doppler US enhancement. Dopler CEUS perameters in that study had 79-100% sensitivity of 79-100% and 77-92% specificity (20). Marret et al reported that washout times and areas under the curves were significantly greater in ovarian malignancies than in other benign tumors (P < .001), leading to sensitivity estimates between 96% and 100% and specificity estimates between 83 and 98%. They concluded that Doppler CEUS parameters had slightly higher sensitivity and slightly lower specificity when compared with transvaginal sonographic variables of the resistive index and serum cancer antigen 125 levels (21).

Our clinical studies explored differences in enhancement parameters in benign versus malignant ovarian masses using the new method of CEUS using pulse inversion harmonic imaging (21, 22). This method produces more reliable estimates of tumor microvascular perfusion and provides more consistent results compared to Doppler CEUS. We reported that all malignant tumors and 50% of benign ones showed detectable contrast enhancement (image intensity >10% above the baseline) after contrast injection. When contrast enhancement dynamics were assessed, we found that malignant lesions had a similar time to peak (26.2 ± 5.9 versus 29.8 ± 13.4 seconds; P = .4), greater peak enhancement (21.3 ± 4.7 versus 8.3 ± 5.7 dB; P < .001 ), a longer half wash-out time (104.2 ± 48.1 versus 32.2 ± 18.9 seconds; P < .001), and a greater AUC (1807.2 ± 588.3 versus 413.8 ± 294.8 seconds–1; P < .001) when compared with enhancing benign lesions. Our data suggest that, except for the wash-in time, contrast enhancement parameters are significantly different in benign versus malignant ovarian masses. The wash-in time probably reflects intrinsic circulation depending on cardiac contraction, blood pressure, and overall vascular tone. Once blood circulates through the tumor, however, differences may reflect the unique branching patterns and vessel morphologic characteristics in the microvascularity of the tumors. The area under the enhancement curve greater than 787 seconds–1 was the most accurate diagnostic criterion for ovarian cancer, with 100.0% sensitivity and 96.2% specificity. Additionally, peak contrast enhancement of greater than 17.2 dB (90.0% sensitivity and 98.3% specificity) and a half wash-out time of greater than 41.0 seconds (100.0% sensitivity and 92.3% specificity) proved to be useful. These results show that contrast-enhanced PIH sonography is a more appropriate method for characterizing blood flow dynamics in ovarian tumors, and it can provide an important tool to aid differential diagnoses between benign and malignant ovarian tumors.

In summary, contrast enhancement patterns significantly differ between benign and malignant ovarian masses. The addition of a vascular ultrasound contrast agent allows a more complete delineation of the vascular anatomy through enhancement of the signal strength from small vessels and provides an entirely new opportunity to time the transit of an injected bolus. Contrast sonography has higher sensitivity and specificity to differentiate between benign and malignant lesions than conventional TV-US and for discriminating between endometriosis and detecting occult Stage I disease.

(1) Bazot M, Daraï E, Nassar-Slaba J, Lafont C, Thomassin-Naggara I. Value of magnetic resonance imaging for the diagnosis of ovarian tumors: a review. J Comput Assist Tomogr 2008; 32:712–723

(2) Risum S, Hogdall C, Loft A, et al. The diagnostic value of PET/CT for primary ovarian cancer: a prospective study. Gynecol Oncol 2007; 105:145–149.

(3) Roman LD, Muderspach LI, Stein SM, Laifer-Narin S, Groshen S, Morrow CP. Pelvic examination, tumor marker level, and gray-scale and Doppler sonography in the prediction of pelvic cancer. Obstet Gynecol 1997; 89:493–500.

(4) Reles A, Wein U, Lichtenegger W. Transvaginal color Doppley sonography and conventional sonography in the preoperative assessment of adnexal masses. J Clin Ultrasound 1997; 25: 217-225.

(5) Hamper UM, Sheth S, Abbas FM, Rosenshein NB, Aronson D, Kurman RJ. Transvaginal color Doppler sonography of adnexal masses: differences in blood flow impedance in benign and malignant lesions. AJR Am J Roentgenol 1993; 160:1225–1228.

(6) Liu JH, Zanotti KM. Management of the Adnexal Mass. Obstet Gynecol 2011; 117: 1413-1428.

(7) Hassen K, Ghossain MA, Rousset P, Sciot C, et al. Characterization of Papillary Projections in Benign Versus Borderline and Malignant Ovarian Masses on Conventional and Color Doppler Ultrasound. AJR Am J Roentgenol 2011; 196: 1444- 1449.

(8) Kinkel K, Hricak H, Lu Y, Tsuda K, Filly RA. US characterization of ovarian masses: a meta-analysis. Radiology 2000; 217:803–811.

(9) Fishman DA, Cohen L, Blank SV, et al. The role of ultrasound evaluation in the detection of early-stage epithelial ovarian cancer. Am J Obstet Gynecol 2005;192 : 1214–1221; discussion, 1221–1212

(10) Cohen L, Fishman DA. Ultrasound and ovarian cancer. Cancer Treat Res 2002; 107 : 119–132

(11) Dutta S, Wang FQ, Fleischer AC, Fishman DA. New Frontiers for Ovarian Cancer Risk Evaluation: Proteomics and Contrast-Enhanced Ultrasound. AJR Am J Roentgenol. 2010; 194(2): 349-354.

(12) Yankeelov TE, Niermann KJ, Huamani J, Kim DW, Quarles CC, et al. Correlation Between Estimates of Tumor Perfusion From Microbubble Contrast-Enhanced Sonography and Dynamic Contrast-Enhanced Magnetic Resonance Imaging. J Ultrasound. 2006; 25: 487-497.

(13) Marret H, Sauget S, Giraudeau, Brewer M, et al. Contrast-Enhanced Sonography Helps in Discrimination of Benign From Malignat Adnexal Masses. J Ultrasound Med 2004; 23: 1629-1639

(14) Brasch R, Turetschek K. MRI characterization of tumors and grading angiogenesis using macromolecular contrast media: status report. Eur J Radiol 2000;34 : 148–155

(15) Leen E. Ultrasound contrast harmonic imaging of abdominal organs. Semin Ultrasound CT MR 2001;22 : 11–24

(16) Orden MR, Jurvelin JS, Kirkinen PP. Kinetics of a US contrast agent in benign and malignant adnexal tumors. Radiology2003 ; 226:405 –410

(17) Ferrara KW, Merritt CR, Burns PN, Foster FS, Mattrey RF, Wickline SA. Evaluation of tumor angiogenesis with US: imaging, Doppler, and contrast agents. Acad Radiol 2000;7 : 824–839

(18) Hall GH, Atkin SL, Turnbull LW. Use of dynamic contrast-enhanced MRI to assess the functional vascular pharmacokinetic parameters of normal human ovaries. J Reprod Med 2002;47 : 107–114

(19) Kupesic S, Kurjak A. Contrast-enhanced, three-dimensional power Doppler sonography for differentiation of adnexal masses. Obstet Gynecol 2000; 96:452–458.

(20) Orden MR, Juvenlin JS, Kirkinien PP. Kinetics of a US contrast agent in benign and malignant adnexal tumors. Radiology 2003; 226:405–410.

(21) Marret H. Sauget S, Giraudeau B, et al. Contrast-enhanced sonography helps in discrimination of benign from malignant adnexal masses. J Ultrasound Med 2004; 23:1629–1639.

(22) Fleisher AC, Lyshchik AP, Jones HW, Fishman DA. Early Detection of ovarian cancer with contrast enhanced transvaginal sonography. 2009; 53:49-54

Detection of early stage EOC is difficult. The following innovative sonographic techniques are currently used to improve identification of early stage EOC.

Ultrasound Contrast Imaging - Contrast-enhanced sonography is a great tool for detection and characterization of angiogenesis. Ultrasound contrast agents for intravenous use consist of small, stabilized microbubbles usually on the order of 1-10 microns in diameter (1). These bubbles cause increased echogenicity and are thus termed, echoenhancers. The agents create this effect by causing an acoustic impedance mismatch with the adjacent tissues. This, in turn, causes increased scattering and reflection of the sound beam, thereby leading to increased sonographic signal and increased echogenicity. The degree of echo-enhancement depends on a multitude of factors including the size of the microbubble, the concentration of contrast agent, and the compressibility of the bubbles as well as the interrogating ultrasound frequency (1).

Pulse Inversion Harmonic Imaging (PIH) - In Pulse Inversion Harmonic imaging two pulses are transmitted down each ray line. The first is a normal pulse, the second is an inverted replica of the first so that wherever there was a positive pressure on the first pulse there is an equal negative pressure on the second. Any linear target such as soft tissues responds equally to positive and negative pressures will reflect back to the transducer equal but opposite echoes. Microbubbles respond in non-linear fashion and do not reflect identical inverted waveforms. This allows the separation of the fundamental component of the bubble echoes from the background, improving resolution, and increasing sensitivity to contrast agents.

Microvascular Imaging (MVI). Pulse Inversion Harmonic Imaging has led to the ability to image individual bubbles in small vessels within and adjacent to tumors with very low blood flow rates. MicroVascular Imaging allows to capture and track the bubbles as they go around and through these small leaky vessels, providing improved visualization of slowly perfused tumors. The MIP technique involves selection of maximum pixel values throughout consecutive, PIH images as the bubbles enter of replenish replenish the imaging plane. A composite image showing the vascular architecture is constructed and can be used to improve aour abitily to detect areas of abnormal vascularity.

Flash Contrast Imaging While the ability to visualize microvascular blood flow in real-time is a significant advancement, the ability to destroy contrast at will, also has diagnostic potential. By destroying the contrast within the scanplane, a “negative bolus” of contrast is created locally. Then, the time it takes for contrast to refill the scan plane and the amount of the contrast in the ROI may be used for estimation of microvessel cross-sectional area, blood flow velocity and tumor perfusion (2).

Contrast enhanced sonography may significantly improve the diagnostic ability of ultrasound to identify early microvascular changes that are known to be associated with early stage ovarian cancer (1, 3). Currently, contrast agents play a pivotal role in the imaging modalities of computed tomography (CT) and magnetic resonance imaging (MRI) by increasing image conspicuity. By increasing the density or signal intensity of a particular organ and thus, the signal to noise ratio, contrast agents help to detect and characterize parenchymal lesions. Indeed, contrast agents have received such widespread acceptance, that a CT exam performed without intravenous contrast for many indications is now considered limited. Our preclinical studies demonstrated that the intravenous contrast agents in ultrasound holds great promise in a multitude of potential clinical applications, especially in identifying aberrant vascular changes associated with malignancy (1, 4-8).

Previous studies have addressed the use of contrast-enhanced sonography for benign and malignant tumors by showing greater enhancement of malignant tumors on Doppler imaging. According to Kupesic et al the use of a contrast agent with three-dimensional power Doppler sonography showed diagnostic efficiency (95.6%) that was superior to that of nonenhanced three-dimensional power Doppler sonography (86.7%) (9). However, simple documentation of tumor enhancement may not be sufficient because some benign tumors show detectable contrast enhancement. This limitation can be addressed by assessment of the contrast enhancement kinetics. Only 2 studies have been published that used kinetic parameters of the contrast agent to compare benign with malignant tumors in the power Doppler mode. Ordén et al demonstrated that after microbubble contrast agent injection, malignant and benign adnexal lesions behave differently in degree, onset, and duration of Doppler US enhancement. Dopler CEUS perameters in that study had 79-100% sensitivity of 79-100% and 77-92% specificity (10). Marret et al reported that washout times and areas under the curves were significantly greater in ovarian malignancies than in other benign tumors (P < .001), leading to sensitivity estimates between 96% and 100% and specificity estimates between 83 and 98%. They concluded that Doppler CEUS parameters had slightly higher sensitivity and slightly lower specificity when compared with transvaginal sonographic variables of the resistive index and serum cancer antigen 125 levels (11).

Our clinical studies explored differences in enhancement parameters in benign versus malignant ovarian masses using the new method of CEUS using pulse inversion harmonic imaging (11, 12). This method produces more reliable estimates of tumor microvascular perfusion and provides more consistent results compared to Doppler CEUS. We reported that all malignant tumors and 50% of benign ones showed detectable contrast enhancement (image intensity >10% above the baseline) after contrast injection. When contrast enhancement dynamics were assessed, we found that malignant lesions had a similar time to peak (26.2 ± 5.9 versus 29.8 ± 13.4 seconds; P = .4), greater peak enhancement (21.3 ± 4.7 versus 8.3 ± 5.7 dB; P < .001 ), a longer half wash-out time (104.2 ± 48.1 versus 32.2 ± 18.9 seconds; P < .001), and a greater AUC (1807.2 ± 588.3 versus 413.8 ± 294.8 seconds–1; P < .001) when compared with enhancing benign lesions. Our data suggest that, except for the wash-in time, contrast enhancement parameters are significantly different in benign versus malignant ovarian masses. The wash-in time probably reflects intrinsic circulation depending on cardiac contraction, blood pressure, and overall vascular tone. Once blood circulates through the tumor, however, differences may reflect the unique branching patterns and vessel morphologic characteristics in the microvascularity of the tumors. The area under the enhancement curve greater than 787 seconds–1 was the most accurate diagnostic criterion for ovarian cancer, with 100.0% sensitivity and 96.2% specificity. Additionally, peak contrast enhancement of greater than 17.2 dB (90.0% sensitivity and 98.3% specificity) and a half wash-out time of greater than 41.0 seconds (100.0% sensitivity and 92.3% specificity) proved to be useful. These results show that contrast-enhanced PIH sonography is a more appropriate method for characterizing blood flow dynamics in ovarian tumors, and it can provide an important tool to aid differential diagnoses between benign and malignant ovarian tumors.

In summary, contrast enhancement patterns significantly differ between benign and malignant ovarian masses. The addition of a vascular ultrasound contrast agent allows a more complete delineation of the vascular anatomy through enhancement of the signal strength from small vessels and provides an entirely new opportunity to time the transit of an injected bolus. Contrast sonography has higher sensitivity and specificity to differentiate between benign and malignant lesions than conventional TV-US and for discriminating between endometriosis and detecting occult Stage I disease.

(1) Dutta S, Wang FQ, Fleischer AC, Fishman DA. New Frontiers for Ovarian Cancer Risk Evaluation: Proteomics and Contrast-Enhanced Ultrasound. AJR Am J Roentgenol. 2010; 194(2): 349-354.

(2) Yankeelov TE, Niermann KJ, Huamani J, Kim DW, Quarles CC, et al. Correlation Between Estimates of Tumor Perfusion From Microbubble Contrast-Enhanced Sonography and Dynamic Contrast-Enhanced Magnetic Resonance Imaging. J Ultrasound. 2006; 25: 487-497.

(3) Marret H, Sauget S, Giraudeau, Brewer M, et al. Contrast-Enhanced Sonography Helps in Discrimination of Benign From Malignat Adnexal Masses. J Ultrasound Med 2004; 23: 1629-1639

(4) Brasch R, Turetschek K. MRI characterization of tumors and grading angiogenesis using macromolecular contrast media: status report. Eur J Radiol 2000;34 : 148–155

(5) Leen E. Ultrasound contrast harmonic imaging of abdominal organs. Semin Ultrasound CT MR 2001;22 : 11–24

(6) Orden MR, Jurvelin JS, Kirkinen PP. Kinetics of a US contrast agent in benign and malignant adnexal tumors. Radiology2003 ; 226:405 –410

(7) Ferrara KW, Merritt CR, Burns PN, Foster FS, Mattrey RF, Wickline SA. Evaluation of tumor angiogenesis with US: imaging, Doppler, and contrast agents. Acad Radiol 2000;7 : 824–839

(8) Hall GH, Atkin SL, Turnbull LW. Use of dynamic contrast-enhanced MRI to assess the functional vascular pharmacokinetic parameters of normal human ovaries. J Reprod Med 2002;47 : 107–114

(9) Kupesic S, Kurjak A. Contrast-enhanced, three-dimensional power Doppler sonography for differentiation of adnexal masses. Obstet Gynecol 2000; 96:452–458.

(10) Orden MR, Juvenlin JS, Kirkinien PP. Kinetics of a US contrast agent in benign and malignant adnexal tumors. Radiology 2003; 226:405–410.

(11) Marret H. Sauget S, Giraudeau B, et al. Contrast-enhanced sonography helps in discrimination of benign from malignant adnexal masses. J Ultrasound Med 2004; 23:1629–1639.

(12) Fleisher AC, Lyshchik AP, Jones HW, Fishman DA. Early Detection of ovarian cancer with contrast enhanced transvaginal sonography. 2009; 53:49-54

Unfortunately, pelvic examinations are not efficient in distinguishing an early or premalignant lesion from a normal ovary based on palpation (1). Evidence from a screening study at the Duke Evidence-based Practice Center (EPC) under contract to the Agency for Healthcare Research and Quality (AHRQ), demonstrated that the sensitivity and specificity of detecting a pelvic mass based solely on a pelvic examination is 40% and 90%, respectively (2). The same study reported that a pelvic examination is able to distinguish between a benign and malignant mass with 58% sensitivity and 98% specificity (2). The low sensitivity percentages of 40% and 58% illustrate that a pelvic examination may very likely produce a false negative with serious medical consequences. This false negative result may overlook a pelvic mass or it may incorrectly diagnose an individual as not having a malignant tumor. Similarly, the specificity rates of 90% and 98%, which are below the required specificity for an effective screening test, are inefficient because of the likelihood of a false-positive result being produced. A false-positive result may either suggest a pelvic mass that does not actually exist, or diagnose an individual as having a malignant tumor when in actuality the tumor is benign.

(1) Cragun JM. Screening for ovarian cancer. Cancer Control. 2011; 18(1): 16-21

(2) Myers ER, Bastian LA, Havrilesky LJ, Kulasingam SL, Terplan MS, Cline KE, Gray RN, McCrory DC. Management of Adnexal Mass. Evidence Report/Technology Assessment No. 130 (Prepared by the Duke Evidence-based Practice Center under Contract No. 290-02-0025.) AHRQ Publication No. 06-E004. Rockville, MD: Agency for Healthcare Research and Quality. February 2006.

Pelvic examination, transvaginal ultrasound, and serum CA125 level is the current the standard in screening for ovarian cancer. As this multimodal method is not without flaws, efforts are underway to strengthen its screening ability, or find better methods altogether.

The issue with CA125 as a tumor marker is that changes in its serum levels are not specific to ovarian cancer. Elevations may be physiologic as in ovulation or pathophysiologic as in fibroids, endometriosis in addition to ovarian cancer (4). In screening for ovarian cancer the specificity of serum CA125 level is higher in postmenopausal women. In screening for early stage ovarian cancer, CA125 has not been found to be very useful due to a decrease in sensitivity with that stage of disease. CA-125 is often used as a component for the evaluation of a pelvic mass, but it is nonspecific to ovarian cancer. CA-125 is elevated in many disease states, benign diseases, and physiological conditions (1). Elevated CA-125 levels may be caused by: pancreatic, breast, bladder, liver, and lung malignancies, ectopic pregnancy, benign ovarian cysts, endometriosis, uterine fibroids, ovulation, pregnancy, and menstruation (3). Since elevated CA-125 levels are nonspecific to just ovarian cancer, there is a possibility that using CA-125 as an assay to diagnosis ovarian cancer can result in a number of false-positive results. It has been noted that because CA-125 levels are naturally elevated due to ovulation, endometriosis, and other benign conditions, CA-125 as a tumor marker is more effective in postmenopausal women (5). CA-125 is only elevated in 47% of women with early-stage ovarian cancer, while CA-125 levels are elevated in 80% to 90% of advanced-stage ovarian cancers. (2, 3). Furthermore, CA-125 has a sensitivity of only 50% to 60% for early-stage diagnosis in postmenopausal women, when specificity is set at 99% (5). Clinically CA-125 is useful to follow women diagnosed with ovarian cancer for prognosis, surveillance, and optimization of care.

(1) Cass L, Karlan BY. Ovarian Cancer Speak Out-But What Are They Really Saying? J Natl Cancer Ist. 2010; 102(4): 211-212

(2) Dutta S, Wang FQ, Fishman DA. The dire need to develop a clinically validated screening method for the detection of early-stage ovarian cancer. Biomark Med. 2010; 4(3): 437-439.

(3) Dutta S, Wang FQ, Phalen A, Fishman DA. Biomarkers for ovarian cancer detection and therapy. Cancer Biol Ther. 2010; 9(9): 668-677

(4) Rein BJD, Gupta S, Dada R, Safi J, Michener C, Argawal A. (Review Article) Potential markers for detection and monitoring of ovarian cancer. Journal of oncology. Vol 2011

(5) Yurkovetsky Z, Skates S, Lomakin A, Nolen B, Pulsipher T, Modugno F, Marks J, Godwin A, Gorelik E, Jacobs I, Menon U, Lu K, Badgwell D, Bast RC Jr, Lohkhin AE. Development of a multimarker assay for early detection of ovarian cancer. J Clin Oncol. 2010; 28(13): 2159-2166.

CA125 is physiologically expressed in epithelial tissues. Examples of these tissues include those of mullerian origin such as endometrium, endocervix, and fallopian tubes, those of coelomic origin such as pleural mesothelium, pericardial, and peritoneal. CA125 has also been found in conjunctiva (1, 3). It is elevated in various conditions including endometriosis, pregnancy, uterine leiomyomas, benign and malignant ovarian tumors, liver disease, cancers of the stomach, pancrease, colon, uterus and fallopian tubes (2). Free CA125 is found after the extracellular domain is cleaved near the cell surface membrane (3). The antigen was also found outside the cell, in body fluids like seminal fluid, and culture media. One author quoted CA 125’s sensitivity for ovarian cancer as being 52% (4). A study by Nakae et al demonstrated 66.3% specificity and 84.4% sensitivity for CA125. As this has been the oldest and one of the best performing biomarkers, it is likely that a biomarker panel used to detect ovarian cancer in its early stage will include CA-125 (5).

(1) Dutta S, Wang FQ, Fishman DA. The dire need to develop a clinically validated screening method for the detection of early-stage ovarian cancer. Biomark Med. 2010; 4(3): 437-439.

(2) Dutta S, Wang FQ, Fleischer AC, Fishman DA. New Frontiers for Ovarian Cancer Risk Evaluation: Proteomics and Contrast-Enhanced Ultrasound. AJR Am J Roentgenol. 2010; 194(2): 349-354.

(3) Bast RC, Spriggs, DR. (Editorial) More than a biomarker: CA125 may contribute to ovarian cancer pathogenesis. Gynecologic oncology; 121 (2011): 429-430

(4) Rein BJD, Gupta S, Dada R, Safi J, Michener C, Argawal A. (Review Article) Potential markers for detection and monitoring of ovarian cancer. Journal of oncology. Vol 2011

(5) Drapkin D, Adam C, Skates S. uPar: a beacon of malignancy? Clinical Cancer Research. 2008 September 15; 14(18): 5643-5645

While dozens of potential serum markers have been identified, CA-125 is the most thoroughly assessed and most frequently used biomarker. CA-125, which was first developed in 1981, is a high-molecular-weight glycoprotein originally detected by a murine monoclonal antibody (OC125). CA-125 is a coelomic epithelial antigen produced by mesothelial cells that line the peritoneum, pleural cavity, and pericardium (1). The 1970’s marked the discovery of CA125. Monoclonal antibodies to that protein were created by immunizing mice with human ovarian cancer cells, known as AVCA433, resecting their spleens and creating hybridomas. The appropriate antibody clones produced by these immunized mice were selected according to their affinity for ovarian cancer cells and lack thereof for B-lymphocytes that were made immortal by infection with Epstein Barr Virus. The one hundred twenty fifth antibody clone was interesting in that it showed specificity to the endometrium and fallopian tube, in addition to ovarian cancer cells but would not bind to normal ovarian epithelium. This antibody was named Ovarian Cancer 125 and the target it recognized was Cancer Antigen 125 (75).

Structurally, CA125 is a mucin-like high-molecular weight glycoprotein with a molecular weight ranging from 2.5 to 5 million Dalton (2,3). It is a type I protein and is like other mucins in that the molecule spans the cell membrane. CA125 has an N-terminal extracellular domain made up of more than 12000 amino acids that is greatly glycosylated. This domain has a tandem repeat portion. The C-terminal domain of CA125 is a cytoplasmic tail accepted as having the potential to be phosphorylated during signal transduction (3). It is encoded by the MUC16 gene found on chromosome 19p13.2 (2). CA125 is expressed in 80% of ovarian cancers it is amplified in only 5% and mutated in 4.7% of high-grade ovarian cancers (2). Because of this, it is thought that of CA125 expression is regulated at a transcriptional or post-transcriptional level (2). Shedding and/or expression of CA125 is highly regulated and expression is modestly increased by inhibition of PKC beta, tyrosine phosphatase, and EGFR as well as by stimulation of PKA. Expression is moderately decreased by decreased levels of EGF and LPA while glucocorticoids cause a significant drop. Agents not shown to have any effect on CA125 expression or shedding include IGF, estrogen, IFN-alpha and gamma, TNF, VEGF, M-CSF, and IL-6 (2).

The function of CA125 is suggested by its interaction with other proteins. For example CA125 selectively binds to mesothelin and possibly leads to peritoneal adhesion of ovarian cancer cells (2, 3). By virtue of its glycosylated portion CA125 can also bind to galectin which allows cancer cell adhesion to other tissues (2). Various genetic experiments have contributed to the understanding of CA125’s function. In female mice MUC16 is thought to occupy the same locations as in humans. This being the case genetically altered mice have been used to better understand the function of CA125/MUC16. SKOv3 ovarian cancer cells with cell surface MUC 16 genes knocked down were constructed and have decreased interval stationary growth, decrease in clonogenic growth, and inhibition of xenografic growth. On the other hand, adding the MUC16 construct that was previously knocked-down creates an aggressive line of cancer cells as it leads to enhancement of anchorage dependent cell growth, cell migration, colony formation independent of anchorage, invasion, and metastasis (2). In addition these cells showed evidence of transition from epithelial to mesenchymal tissue types via up-regulation of N-cadherin and vimentin expression and down-regulation of E-cadherin expression (2). Further evidence of CA125’s function comes from a reduction in the potential for invasion and adhesion when CA125 RNAi is knocked-down in SKOV-8 cells (2). Lastly CA125 may inhibit the activity of NK cells by precluding the synapse with ovarian cancer cells. As the above observations would support poor outcomes are associated with elevated CA125 levels. This is not dependent on tumor burden. Additionally lack of CA125 expression at higher stages is a poor prognostic sign.

(1) Moore RG, MacLaughlan S. Current clinical use of biomarkers for epithelial ovarian cancer. Curr Opin Oncol. 2010; 22(5): 492-497.

(2) Bast RC, Spriggs, DR. (Editorial) More than a biomarker: CA125 may contribute to ovarian cancer pathogenesis. Gynecologic oncology; 121 (2011): 429-430

(3) Theriault C, Pinard M, Comamala m, Migneault M, beaudin J, Matte I, Boivin M, Piche A, Rancourt C. MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis. Gynecologic oncology 121 (2011) 434-443.

Clinically CA-125 is useful to follow women diagnosed with ovarian cancer for prognosis, surveillance, and optimization of care.