Screening for ovarian cancer: imaging challenges and opportunities for improvement.

Abstract

When ovarian cancer is detected at an early, localized stage (Stage I or II), cytoreductive surgery – currently the most effective treatment – and conventional chemotherapy can cure 70–90% of patients, compared with approximately 20% or fewer when it is diagnosed at later stages (Stages III and IV)1. Even aggressive high-grade, poorly differentiated or undifferentiated tumors have a much higher 5-year survival rate when diagnosed in Stage I/II rather than Stage III/IV (74% vs 27%)2. Clearly, early staging is critical, yet only 20–25% of ovarian cancers are diagnosed early. One option for reducing risk in high-risk women is through prophylactic bilateral salpingectomy or salpingo-oophorectomy; however, these procedures are inappropriate for normal-risk women, who represent 75–85% of ovarian cancer cases3-5. In light of these statistics, efforts have been made to develop effective ovarian cancer screening strategies. In this Editorial, we conduct a critical review of current ovarian cancer screening approaches, focusing on the role of ultrasound, including its strengths and weaknesses, as well as additional strategies, such as implementation of a three-stage multimodal approach, to potentially improve screening in the future. Ovarian masses are classified pathologically as benign, malignant (invasive) or of low malignant potential (borderline). Borderline tumors, which do not invade the basement membrane underlying the epithelium and have markedly atypical histology, account for approximately 15% of epithelial ovarian tumors and generally have a much better prognosis than do invasive ovarian tumors, with 10-year survival rates ranging from 85% to 98%, depending on histologic type, disease stage and patient age6, 7. Among invasive ovarian cancers there are two major subtypes, often classified as Type-I or Type-II tumors8, 9. Type-I tumors typically present in the early stages and are clinically indolent; they include low-grade serous carcinomas, low-grade endometrioid carcinomas, mucinous carcinomas, clear-cell carcinomas and malignant Brenner (transitional) tumors. Type-II tumors account for approximately 75% of epithelial ovarian cancers and include high-grade serous carcinomas, high-grade endometrioid carcinomas, undifferentiated carcinomas and malignant mixed mesodermal tumors. High-grade serous carcinoma is the most common morphology, accounting for 50–70% of invasive ovarian cancers. Most, if not all, Type-II tumors have TP53 mutations and are very aggressive, and typically they present in the late stages10, 11. Because Type-I tumors are generally less aggressive, they are more likely to grow slowly to a large size while remaining within the ovaries, and thus are more likely to be detected at an early stage. In contrast, aggressive Type-II ovarian tumors often metastasize before detection, with Stage-I high-grade serous ovarian cancer representing only 1% of primary ovarian cancer diagnoses using conventional methods12, 13. A retrospective chart review comparing the average size of primary ovarian tumors across early-stage (I/II) and late-stage (III/IV) patients further supports the theory of two distinct ovarian cancer subtypes by revealing significantly larger sizes among early-stage cases (average diameter, 10.7 cm vs 4.8 cm)14. Type-I tumors are believed to originate from precursor lesions in the ovary (e.g. endometriosis or borderline tumors), while Type-II tumors are thought to develop de novo from the ovarian surface epithelium, subserosal inclusion cysts or from the fimbriae of the Fallopian tubes15-17. Mouse models support development of high-grade serous ovarian cancer from both the Fallopian tubes and the ovaries18-20. Samples collected during prophylactic salpingo-oophorectomies performed on women with BRCA1 or BRCA2 germline mutations have revealed tubal involvement in an estimated 76% of early gynecologic malignancies21-26. As 10–15% of invasive ovarian cancers arise in BRCA1/2-mutation carriers, at least 10% of all ovarian cancers arise from the Fallopian tubes27, 28. Combining this statistic with that of sporadic (non-familial) high-grade serous carcinomas that coat the ovary rather than growing from the surface and likely arise from the Fallopian tubes, which represent approximately 20% of all cases, suggests that at least 30% of ovarian cancers may originate from the Fallopian tubes29-31. Examination of Fallopian tube specimens has revealed high expression of p53 and clonality between serous tubal intraepithelial carcinomas and high-grade serous ovarian carcinomas32-35. Cells in the distal region of the Fallopian tubes are likely more prone to malignant transformation due to the proinflammatory microenvironmental factors associated with ovulation, as well as the relatively large surface area of the fimbriae. Once serous tubal intraepithelial carcinoma develops, these malignant cells are believed to migrate onto the nearby ovarian surface and/or the surrounding peritoneum. Metastatic cells are transported through the peritoneal fluid and implant on the surface of the omentum or the visceral or parietal peritoneum, which provide a favorable microenvironment for growth of cancer cells36-38. Both primary ultrasound screening and multimodal strategies incorporating ultrasound have been evaluated for ovarian cancer detection in several large-scale clinical trials. The University of Kentucky Ovarian Cancer Screening Project screened with transvaginal sonography (TVS) 37 293 women annually between 1987 and 2011; to reduce false positives, measurements of the serum biomarker CA125 were also taken into account after detection of a pelvic mass39. The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial evaluated a non-sequential, multimodal strategy, which employed annual TVS and annual screening for single elevated values of serum CA125. The PLCO trial recruited 78 216 women between the ages of 55 and 74 to either undergo annual ovarian cancer screening (n = 39 105) or to receive conventional care (n = 39 111)40. Because the two screening modalities were conducted independently and not used in combination, referral to a gynecologist resulted either from abnormal TVS findings or from an elevated CA125 measurement. The United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), which enrolled postmenopausal women at average risk of ovarian cancer, evaluated both a sequential, multimodal arm and a primary ultrasound arm41. In this trial, 101 359 women received conventional care as controls, 50 639 underwent annual ultrasound examinations, and 50 640 underwent annual CA125 measurements, which were analyzed using a risk of ovarian cancer algorithm (ROCA)42 in which rising CA125 levels, even if within the normal range, prompted ultrasound examination. In the multimodal screening arm, rising CA125 levels prompted TVS in approximately 2% of participants each year. For a screening strategy to be considered effective, it must achieve a sufficient positive predictive value (PPV). Achieving a high PPV is important for reducing unnecessary operations and the risk of surgery-related complications in otherwise healthy women. In the UKCTOCS, 3.4% of false-positive screens (i.e. women who underwent surgery and were diagnosed with normal or benign pathology) experienced surgery-related complications (e.g. hollow viscus injury, hemorrhage, hernia, wound breakdown, infection)43. In the PLCO trial, a much higher surgery-related complication rate of 15% was reported across screened women who had normal or benign pathology (false-positive screens)44. As one might expect, in both the UKCTOCS and PLCO trials, more women underwent unnecessary operations in the screening group compared with in the usual care group43, 44. There is a consensus that a minimum PPV of 10% is required, such that no more than 10 operations are performed for each ovarian cancer diagnosis45. In addition to having sufficiently high sensitivity and specificity to achieve an adequate PPV, an effective strategy must also reduce mortality in a cost-effective manner46. The University of Kentucky Ovarian Cancer Screening Project reported a PPV of 14.5% and a statistically significant stage shift, with 70% of screen-detected invasive epithelial ovarian cancers being in early Stage I or II39. Across all screen-detected invasive epithelial ovarian cancers, the mean ± SD 5-year survival rate was 74.8% ± 6.6% compared with 53.7% ± 2.3% for unscreened patients treated at the same facility following the same clinical practices. Because survival rates, which are subject to lead-time bias, were reported rather than mortality, these results do not, however, prove that screening reduced deaths from the disease, and the perceived survival benefit may instead be attributable to the earlier diagnosis of disease, with no impact on lifespan. Early results from the prevalence phase of the UKCTOCS were encouraging, suggesting that sequential, multimodal screening (i.e. CA125 with ROCA followed by ultrasound) was able to detect 89.5% of all primary invasive epithelial ovarian and tubal cancers, with a specificity of 99.8% and a PPV of 35.1% (i.e. three operations for each ovarian cancer diagnosis)41. Furthermore, 47% of prevalent ovarian cancers detected through screening were early stage. A smaller study of 4675 women, coordinated by the MD Anderson Specialized Program of Research Excellence (SPORE) in Ovarian Cancer, which utilized the same screening approach as the multimodal arm of the UKCTOCS, reported a similar specificity of 99.9% and a PPV of 40%47. Mortality results from the UKCTOCS were also encouraging, with a significant reduction of 20% in the average overall mortality among patients who underwent sequential, multimodal screening (excluding prevalent cases and primary peritoneal disease); however, given the broad confidence limits around the estimate of reduced mortality, additional follow-up is needed before final conclusions can be drawn regarding the survival advantage of such screening43. Comparison of results from the primary ultrasound and multimodal screening arms of the UKCTOCS revealed a lower sensitivity for detecting invasive epithelial ovarian and Fallopian tube cancers diagnosed within 1 year of screening, a lower percentage of early-stage (Stage I/II) invasive ovarian, tubal and undesignated cancers, and a lower PPV for detecting invasive ovarian and tubal cancers in the ultrasound screening arm (66%, 23%, and 5% vs 87%, 38% and 23%, respectively)43. Furthermore, the ultrasound arm exhibited a smaller reduction in mortality of 11–12% (including prevalent cases), compared with 15–16% in the multimodal arm. In contrast to the promising results obtained in the UKCTOCS, which suggest a high PPV, a shift to earlier stage diagnosis and a possible survival benefit, the screening strategy evaluated in the PLCO failed to improve survival or to detect an increased fraction of early-stage ovarian cancer cases44. Additionally, the PPV for detecting invasive ovarian cancers was much lower in the first four annual screening rounds of the PLCO than in the UKCTOCS, at 2.6% or 0.9% for women with a positive CA125 or TVS screen, respectively48. In retrospect, the PPV would have risen to 20% if both CA125 and TVS screens were positive, although over 80% of cases would have been missed. Possible explanations for the discrepancy between the PLCO and the UKCTOCS findings could be that a non-sequential approach was used in the PLCO (i.e. TVS and CA125 screens were conducted independently of one another). Additionally, the PLCO trial employed a fixed cut-off for CA125, whereas the UKCTOCS utilized each woman's own baseline and prompted referral for TVS even among women with CA125 levels below an arbitrary cut-off defined for the general population. Furthermore, in the UKCTOCS, women with a modest rise in CA125 and an intermediate risk of developing ovarian cancer were retested 3 months later, while, in the PLCO trial, women returned after 1 year. Another explanation for the absence of a mortality benefit in the PLCO trial could lie in the lack of a protocol for managing patients with positive screens. Management was instead left to the discretion of each patient's physician, without a clear policy for follow-up and surgical exploration49. Because prompt surgical intervention, optimal cytoreductive surgery and effective chemotherapy each impact survival, if physicians were unsure as to how to manage a positive screen or preferred to take a watchful waiting approach, a long lapse between an initial positive screen and surgery could have resulted, negating the expected benefit of screening and minimizing the chance of producing a stage shift. While the inclusion of specified protocols mandating patient management following positive and negative screens was a strength of the UKCTOCS, one potential disadvantage experienced by some patients in the multimodal arm was increased time to surgical intervention, given the need for repeat testing41. Among women in the multimodal arm who were diagnosed with ovarian or tubal malignancies during the prevalence phase of the UKCTOCS, 21% were characterized as intermediate risk during the Level-1 screen, which called for additional follow-up testing, extending the median time to surgery to 273.9 days. A different protocol for triaging patients was followed for the ultrasound arm, in which the median time from the Level-1 scan to surgery was 81.5 days for screen-detected ovarian or tubal malignancies41. Although early-stage ovarian tumors are believed to double in volume every 4 months, the relevance of an extended time from detection to surgery and its effect (if any) on lifespan is unknown in this case50. TVS is generally accepted as the primary imaging modality for evaluation of an adnexal mass and has largely replaced transabdominal ultrasound given its detailed anatomic depiction of pelvic anatomy, superior resolution and better performance in obese patients51-53. TVS is an attractive screening modality because it is safe, cost-effective and well-tolerated by patients. When included as part of a multimodal ovarian cancer screening strategy based on serum biomarkers (e.g. CA125), the primary benefit of ultrasound is to reduce false positives and therefore the number of unnecessary operations. Inclusion of ultrasound can also be useful in diagnosing cases in which expression of CA125 is weak or absent, which has been shown to be the case in up to 22% of ovarian cancer tissue samples54. Some benign ovarian and gynecologic conditions (e.g. benign ovarian cysts, endometriosis, leiomyoma) cause elevated serum CA125 levels, resulting in false positives55, 56. Thus, the PPV of serum CA125 is insufficient to warrant its use as a stand-alone ovarian cancer screening modality. In the first four screening rounds of the PLCO, a PPV of 2.6% was achieved when using single serum CA125 levels at or above a fixed threshold of 35 U/mL, while a much higher PPV of 20% was reported when positive CA125 and TVS results were combined retrospectively48. As an improvement over using a fixed CA125 cut-off, applying the ROCA to CA125 measurements collected serially from over 9000 women at Royal London and St. Bartholomew's Hospitals achieved a PPV of 15%42. However, despite showing improvement over a threshold approach, the PPV of the ROCA approach is still inferior to that of multimodal screening (ROCA followed by TVS), which had a 23% PPV in the UKCTOCS43. In addition to improving the PPV of ovarian cancer screening, another potential benefit of including TVS is that physicians may be more confident and trusting of abnormal TVS results than of abnormal CA125 results: physicians in the PLCO trial were more likely to refer to surgery based on a positive ultrasound screen than on a positive serum CA125 screen. In four rounds of annual screening in the PLCO trial, the surgery rate following a positive TVS screen was 27.2%, while the surgery rate following a positive CA125 screen was 11.7%; of note, while the overall surgery rates among those with positive screens dropped after the first year, it remained higher for TVS than for CA125 in all 4 years48. TVS enables high-resolution imaging of the ovaries and is especially useful in distinguishing simple cysts from complex cystic masses and solid tumors, which is important given the ultrasound characteristics of histologically proven adnexal malignancies. Because the features indicative of malignancy, including cystic lesions with a large solid component, thick wall and/or septations greater than 3 mm in thickness, mural nodules and necrosis of a solid component/mass, are the same for TVS and other imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI), these modalities offer limited additional value for ovarian cancer screening. Furthermore, the spatial resolution of ultrasound generally exceeds that of CT and MRI in diagnosing small lesions. Because CT offers a comparable ability to detect malignancy, yet has a higher cost and exposes patients to ionizing radiation, it is not recommended for ovarian cancer screening57. Positron emission tomography (PET) with CT (PET/CT) is also not recommended for ovarian cancer screening, since it has relatively low spatial resolution, which hinders the detection of small tumors, and involves exposure to ionizing radiation. Additional challenges of using PET/CT include physiologic uptake in normal structures, such as ovarian follicles in the late follicular to early luteal phase in premenopausal women, adjacent bowel mucosa and excretion into the bladder, which may obscure small pelvic malignancies, as well as other confounding conditions that may lead to false-positive (inflammation, endometriosis, pedunculated leiomyomas) and false-negative (early adenocarcinomas, borderline/low-grade tumors) results57. A wide range of sensitivity and specificity (58–100% and 67–92%, respectively) has been reported for the detection, using PET or PET/CT, of ovarian malignancies in women with adnexal masses58-61. While the sensitivity and spatial resolution of ultrasound is generally better or comparable to those of other imaging modalities, MRI has been reported to have greater accuracy and specificity in the diagnosis of malignant adnexal masses (89% and 84%, respectively, vs 64% and 40% for ultrasound)62. However, because of its relatively high cost and lower availability, MRI is not typically the first-line imaging modality for evaluation of adnexal masses. While imaging is the focus of this review, it is important to note that the sensitivity of a sequential, multimodal ovarian cancer screening method, such as the one evaluated in the UKCTOCS, is initially limited by the sensitivity of the first-line test (e.g. serum CA125). As such, the overall mortality benefit and cost effectiveness of screening is highly dependent upon the first-line screen63. That being said, second-line screening has the potential to further limit sensitivity, and the lower sensitivity reported in the ultrasound arm compared with the multimodal arm of the UKCTOCS suggests that TVS may hinder early detection. In addition to reporting lower sensitivity in the ultrasound arm, multiple cases of women with ovarian cancer who had rising serum CA125 levels but normal ultrasound screens were documented in the multimodal arm. The biology of ovarian cancer, which is believed to consist of two primary disease subtypes, poses an obstacle in that annual screening with TVS is expected to be more effective in detecting the more indolent, Type-I tumors in the early stages39, 64. Additionally, a significant fraction of high-grade serous ovarian cancers are believed to originate in the fimbriae of the Fallopian tubes as very small tumors before the cancer progresses to an advanced stage, yet there is little to no documented experience in imaging this anatomy. In the UKCTOCS, there were multiple cases in which the serum CA125 levels were rising, but TVS was normal, in women later diagnosed with ovarian cancer (i.e. false negatives). Model-based estimates indicate that the minimum tumor size necessary to secrete sufficient CA125 as to generate a positive biomarker screen (i.e. serum CA125 > 34.11 U/mL) is 116.7 mm3, which corresponds to a 3-mm-diameter spherical tumor65. Based on this estimate, it is unlikely that tumors smaller than 3 mm in diameter would be detected when using serum CA125 with a fixed cut-off of 34.11 U/mL as a first-line screen; however, ROCA (rather than a fixed cut-off) was used in the UKCTOCS, and multiple patients with serum CA125 levels below 35 U/mL were diagnosed with ovarian cancer. Thus, while the minimum tumor diameter necessary to generate a positive screen with ROCA is unknown, it may be possible to detect tumors smaller than 3 mm. Detecting very small tumors with TVS presents a challenge; of patients in the UKCTOCS diagnosed with invasive epithelial ovarian or tubal cancers that were flagged by ROCA but had CA125 levels below 35 U/mL, 41% showed no abnormality on initial TVS, possibly because their tumor volume was small, given that 49% of these patients were diagnosed at an early stage (Stage I or II)66. As a result of the need for repeat testing, the interval from screening to surgery was significantly longer for the low CA125 patients as compared with patients who had CA125 levels above the 35-U/mL threshold (30 vs 12 weeks, respectively). Thus, there is a need for more sensitive imaging in those patients with positive biomarker screens; it has been suggested that improved imaging is the key to a successful ovarian cancer screening program67. Computer-based models estimate the median diameter of early-stage serous ovarian tumors to be < 3 mm in BRCA-positive women and, during the 4.3 years that these tumors are estimated to persist as early stage, it is believed that they are typically smaller than 9 mm in diameter for over 3.8 of those years50. Thus, while there may be a short window of opportunity for TVS detection of early-stage Type-II tumors which have grown to a detectable size but remain localized, this window may be missed with annual, or even semi-annual, TVS screening68. As a further challenge, data collected during prophylactic salpingo-oophorectomies in patients with BRCA1/2 mutations have revealed primary tumor diameters of < 10 mm in multiple late-stage (Stage III/IV) patients21, 69. Thus, some tumors may metastasize before ever reaching a size that is detectable by TVS. TVS also often fails to detect tumors in women with a normal ovarian volume, such as in cases of primary peritoneal cancer that involves the ovarian surface and does not cause ovarian enlargement. Several studies of women at high risk have revealed minimal to no sonographic abnormalities in patients with high-grade serous ovarian cancer, despite many being in an advanced stage of disease70, 71. Among the false-negative ultrasound screens in the University of Kentucky Ovarian Cancer Screening Project (i.e. women diagnosed with ovarian cancer within 12 months of a negative screen), 33% (3/9) had normal-sized ovaries with extraovarian metastases at the time of surgery and 78% were Stage III72. Likewise, in the prevalence phase of the UKCTOCS, all false negatives reported in the ultrasound screening arm were late stage (Stage III/IV). In the PLCO trial, 85% of screen-detected ovarian cancers with negative TVS screens were late stage at detection (Stage III/IV) and 54% of fatal screen-detected cases had negative TVS screens (not including those without TVS results)73. A lower 5-year survival rate in the PLCO among CA125-positive women with negative TVS screens (42% vs 67% for women with positive TVS screens) could indicate that TVS is inadequate for detecting more aggressive, fatal cancers73. The overlap in sonographic features between early cancer and some benign lesions represents another limitation of ultrasound. The low PPV observed in the ultrasound screening arm compared with the multimodal arm of the UKCTOCS (5% vs 23%, respectively) illustrates the relatively high false-positive rate of ultrasound for the evaluation of adnexal masses43; an even lower PPV of 0.9% based on an abnormal TVS screen was reported in the first four rounds of the PLCO trial48. In both the PLCO and The University of Kentucky Ovarian Cancer Screening Project, primary borderline epithelial neoplasms of the ovary were classified as false positives since they are of low malignant potential and are associated with lower mortality rates. However, these borderline tumors were considered true positives in the UKCTOCS and the majority of screen-detected borderline cases (67%) were from the ultrasound arm43. Of all primary ovarian cancers detected through screening, 30% were borderline in the ultrasound arm, while only 12% of those detected through multimodal screening were borderline. Furthermore, 91% of borderline tumors were detected through screening in the ultrasound arm compared with 55% in the multimodal arm. Similarly, in the initial screen of the PLCO trial, all nine non-invasive cystadenomas of low malignant potential (borderline cases) were detected through TVS40. In addition to detecting more borderline tumors, unpublished data from the UKCTOCS indicate that ultrasound is more sensitive for detecting Type-I vs Type-II tumors74. Because borderline and Type-I ovarian tumors have a much lower incidence of mortality compared with Type-II tumors, detecting an increased fraction of these cases is unlikely to have a substantial impact on mortality. Therefore, while ultrasound is capable of detecting early-stage clinically indolent lesions, such detection may reflect overdiagnosis or the detection of disease that would not, ultimately, cause mortality. A well-documented limitation of ultrasound is operator dependence75. While new equipment and transducers have made TVS much simpler to perform, interobserver variation still exists due to insufficient experience or to misunderstanding regarding ovarian physiological anatomy. Sonologists will likely only gain the necessary experience to improve their performance by carrying out large numbers of exams, as well as seeing the same patient repeatedly in a screening setting and comparing current to prior exams, while consistently ensuring meticulous technique. In the ultrasound arm of the UKCTOCS, 6.2% (n = 3005) of women required a repeat scan following an unsatisfactory scan in which one or both ovaries could not be visualized while also not achieving a good view of the iliac vessels41. Common reasons for non-visualization of the ovaries by TVS include obesity, previous gynecological surgery (hysterectomy, unilateral oophorectomy and tubal ligation), increasing age, presumably due to small atrophic ovaries, and ovaries located superiorly beyond the range of the TVS probe76. When the ovaries cannot be visualized with TVS, transabdominal ultrasound should be attempted, although this is unlikely to improve visualization in obese patients. The inclusion of additional blood-based biomarkers besides CA125 (e.g. HE4 or CA72-4) could further enhance the sensitivity of first-line screening for detecting early-stage ovarian cancer77-80. Techniques using proteomic profiles as biomarkers have also been developed for screening, but have not yet proven sufficiently sensitive or reproducible to be used clinically81, 82. Another potential means of improving first-line screening may come through liquid biopsies, which detect circulating tumor DNA (ctDNA) mutations, circulating tumor cells (CTCs), elevations in the overall level of cell-free DNA (cfDNA) and DNA methylation or other epigenetic biomarkers in extracted bodily fluids83-91. Liquid biopsies based on ctDNA, as compared with CTCs, may offer greater sensitivity for early detection in light of some evidence suggesting a higher yield and frequency of ctDNA in liquid biopsy samples92. Studies detecting ctDNA in ovarian cancer patients in the form of genetic alterations have reported a 41% (9/22) detection rate of known mutations using routine liquid Pap smear samples93, a 93% (28/30) detection rate of tumor-specific p53 sequence in the peritoneal wash fluid94, and an 80% (8/10) detection rate for tumor-specific chromosomal junctions in plasma samples95. Because the aforementioned studies included mainly late-stage patients, the utility of these assays for detecting early-stage disease is uncertain. Additionally, the sensitivity of such assays, which are often performed using polymerase chain reaction techniques and thus have an inherent sensitivity limit of 0.01%, may limit their use for early detection given that ctDNA represents only a fraction of total DNA in the sample96. While evidence of detectable concentrations of ctDNA has been reported among patients with localized disease, indicating that such liquid biopsies may prove useful for detecting early-stage cases, there is currently little or no prospective evaluation of their utility in a screening setting92, 97. Furthermore, there is considerable variability in the concentration of ctDNA in liquid biopsy samples across patients even at the same disease stage (e.g. ctDNA concentrations varying from < 1% to > 40% have been reported among late-stage (Stage III/IV) high-grade serous ovarian cancer patients)95, despite the assumption that concentration is related to tumor burden. Further study is needed to characterize this variability, as well as the relationship between disease extent and ctDNA concentration, to determine if early detection is viable. Additional limitations to the use of liquid biopsies in the screening setting are the time and cost currently required to perform such assays, as well as the potential for a lack of specificity, as in the case of screening for TP53 mutations, which have also been observed in healthy patients98. Because such screening