- Email: [email protected]
Cervical-cancer screening beyond the year 2000
Review
Modern cervical cancer screening
Cervical-cancer screening beyond the year 2000 Alex Ferenczy and Eduardo Franco
Evidence-based studies have shown that new techniques for cervical cancer screening have a higher diagnostic yield than conventional cervical cytology (Pap test). Automated screening devices that use liquid-based, thin-layer cytology and human papillomavirus DNA testing are likely to become the standard for routine primary screening for cervical cancer and its precursors in the 21st century. The increased initial expense of the new techniques will most certainly be absorbed by instituting longer intervals for safe primary screening, in both low-risk and high-risk populations. To make modern screening programmes even more effective, we must promote extensive public awareness campaigns about cervical cancer, a preventable disease. Lancet Oncol 2001; 2: 27–32
Despite the undeniable benefit from screening for cervical cancer with the Papanicolaou (Pap test), there is growing evidence of its limitations in terms of overall diagnostic performance. An extensive meta-analysis on the diagnostic accuracy of cervical cytology screening tests by the US Agency for Health Care Policy and Research1 found that only 86 of a total of 939 studies provided meaningful information. These studies used diagnostic standards such as histology or colposcopy and reported data on both sensitivity and specificity. Even more surprising was the finding that of the 86 studies, only three were not affected by verification bias. In these three studies, published between 1992 and 1995, disease was verified by the diagnostic gold standard, histology, in more than 2000 cytology-negative patients. The sensitivity of conventional cytology ranged from 29% to 56% (mean 49%) and specificity from 97% to 100%.1 The rates are similar to those in the meta-analysis by Fahey and colleagues (42% false-negative cytology).2 Falsenegative results have major medical, financial, and, in some parts of the world, legal implications. On the other hand, false-positive results lead to misuse of a substantial amount of health-care funds and generate unnecessary anxiety in women. The two major causes of false-negative cytology are sampling errors – about two thirds of cases. The remaining third are detection errors. False-positive results are mostly due to interpretation errors. In response to the pitfalls of manual cervical cytology, there has been development of high-technology devices with the potential to optimise Pap test screening programmes by reducing the false-negative and false-positive rates. THE LANCET Oncology Vol 2 January 2001
Figure 1. Thin-layer, liquid-based cytology. With this new technique, virtually all the cells collected from the cervix are suspended in the collection vial.
Technology overview Thin-layer, liquid-based cytology
The basic principle and advantage of this technique over the conventional Pap test is that the sample removed from the cervix is not placed on a glass slide but is rinsed from the sampler into a vial (Figure 1), which contains a cellAF is professor of Pathology and Obstetrics and Gynecology, McGill University and the Sir Mortimer B Davis-Jewish General Hospital, Montréal, Québec, Canada. EF is director of the Division of Epidemiology, Department of Oncology, at McGill University. Correspondence: Professor Alex Ferenczy, Department of Pathology, SMBD-Jewish General Hospital, 3755 Côte Ste Catherine Road, Montréal, Québec, Canada H3T 1E2. Tel: + 1 514 340 7526. Fax: +1 514 340 7542. Email: [email protected]
27
For personal use only. Not to be reproduced without permission of The Lancet.
Review
Modern cervical cancer screening
On the basis of those findings, the US Food and Drug Administration (FDA) approved the ThinPrep Pap test (1996) and AutoCyte PREP System (1999), respectively, as significantly superior and equivalent to the conventional Pap smear for the detection of precancerous and cancerous lesions of the cervix. By September 1999, the performance of the ThinPrep Pap test had been evaluated further in 154 872 lowrisk and high-risk women. In these studies, the cells collected from the cervix were placed in the collection vial (direct-to-vial protocol).5 All but one study used histological verification of positive Pap tests. Spectacular increases in the detection of precancerous lesions were observed, in comparison with historical controls tested with conventional Pap smears a year earlier. For example, the rate of detection of high-grade squamous intraepithelial lesions increased by 26% to 233% and that for low-grade lesions ranged from 40% to 331%. In the largest single study, on 59 539 women, there was a 39% decrease in the ratio of borderline or atypical squamous cells of undetermined significance (ASCUS) to low-grade squamous intraepithelial Figure 2. Cervical cytology. (a) Machine-processed thin-layer cervical sample (ThinPrep Pap), which is devoid of obscuring inflammatory and red blood cells, so interpretation is easier and more lesions.5 Most patients with borderline accurate. (b) Conventional Pap smear containing dense, inflammatory exudate that precludes Pap tests in fact have no disease. Any accurate interpretation of possible diagnostic cells. technique that can lower the preserving transport fluid, thus permitting the collection proportion of false-positive findings is most welcome, of virtually all cellular material in an optimum state. In because it has the potential to decrease substantially the the conventional Pap test, at best only 20% of the cells number of unnecessary colposcopic examinations and the are smeared onto the glass slide and the remaining 80% incremental costs for cervical biopsies and endocervical are lost with the discarded collection device. In the curettage. In the same study, the researchers showed a laboratory, the suspended cells are processed by robotic 99.8% reduction in rates of obscuring blood on slides, a preparation devices such as the ThinPrep (CYTYC Corp, 94.3% reduction in rates of obscuring inflammation, and MA, USA) or AutoCyte PREP System (TriPath Imaging, Inc, a 99.3% decrease in cases of poor collection. These findings NC, USA). Obscuring blood and inflammatory cells are are important contributors to better detection of abnormal automatically removed from the sample, which is then cells. In one multicentre European study, with manually transferred by machine in a thin layer onto a 13 mm prepared AutoCyte PREP direct-to-vial samples, there was (AutoCyte PREP) or 22 mm (ThinPrep) circular area of the an 89% reduction in the proportion of cytological slide (Figure 2). preparations that were “unsatisfactory” and a 74% reduction Large-scale prospective studies evaluated the clinical in the “satisfactory but limited by” category, a 79% performance of the ThinPrep Pap test compared with a improvement in the detection of high-grade squamous histological reference standard.1,3 They found sensitivities intraepithelial lesions, and a 43% decrease in the ASCUS of between 0.73 and 0.94 and specificities of 0.58 to 0.76.1 category compared with 88 569 contemporaneously collected The results of masked, multicentre clinical trials involving conventional Pap smears.6 Another key advantage of liquidmore than 16 000 cases (ThinPrep) and 8807 cases based cytology is that the residual cell suspension can be (AutoCyte PREP) of matched ‘split samples’ (ie Pap smear prepared for histology in cytologically equivocal cases, and first, thin-layer Pap test second) showed a significant most importantly, it can be used for panel testing. With the improvement in slide quality and an overall 18% higher ThinPrep system, one of the leading panel tests is related detection rate (ThinPrep) with the thin-layer approach than to detection of human papillomavirus (HPV) and the other with conventional cytology. AutoCyte PREP yielded to detection of Chlamydia trachomatis. Both tests have detection rates similar to the conventional Pap smear.4 been approved by the FDA. 28
THE LANCET Oncology Vol 2 January 2001
For personal use only. Not to be reproduced without permission of The Lancet.
Review
Modern cervical cancer screening
Computer-assisted automated cytology
At present, the only automated scanning system approved by the FDA for quality control and primary screening purposes is the AutoPap screening system (TriPath Imaging). It scans about 200 conventional Pap smears a day by obtaining images with a high-resolution, high-speed video microscope. The images are digitised, and the morphometric algorithms identify and classify both individual cells and the Pap smear as a whole. Automated scanning of manually screened smears with a negative proportion of 10% as part of quality assurance outperformed human review by a factor of between five and seven, and in primary screening mode Figure 3. Computer-assisted classification of abnormal cells. The left shoulder of the bell-shaped (ie for women without symptoms), it curve represents the 25% least abnormal smears or the ‘sort rate’. These smears do not need detected 100% and 96% of the human review because they are negative. The rest of the smears have cellular abnormalities and need to be reviewed manually. Those located in the right shoulder of the curve are hierarchically the invasive cancers and high-grade most abnormal and need to be reviewed by a supervisor in a quality control mode. squamous intraepithelial lesions, respectively.7 In May 1998, the FDA approved the device at a (secondary screening).8 Similarly, in nine independent 25% ‘sort rate’ in low-risk (not attending a hospital clinic) population-based, international, primary screening studies populations of women. In other words, the device identifies on more than 30 000 women, HC-II was uniformly more the 25% of smears that do not need human review because sensitive for detecting cervical cancers and high-grade they are definitely negative. Conversely, the 75% of the precursors (85% – 100% compared with the 40% – 78% rate smears more likely to be abnormal must be manually for conventional cervical cytology). Combination of the screened by the cytotechnologist (Figure 3). Elsewhere, results of cytology and HPV testing leads to 100% detection including Canada, Europe, and Asia, the device is used at a of high-grade squamous intraepithelial lesions.9,10 Rates of sort rate of 50%. The reason for the differential sort rate referral to colposcopy were 12.5% and 7.0% as a result of approval is that the manufacturer’s submission for approval positive HPV DNA tests and conventional cytology, in the USA preceded the submissions in other countries by respectively. In a population-based primary screening study about a year; by the time of those submissions, further on 2988 women aged 35 years or older, the rate of Papfavourable data had accrued on the diagnostic accuracy of negative HPV-positive women (HPV DNA carriers) was only 4.8% at the recommended cut-off of 1 pg/mL.9 the AutoPap screening device. HPV DNA testing
Prospects
So far, the only HPV test approved by the FDA for commercial clinical use is the Hybrid Capture-II microtitre assay (HC-II/Digene Corp, MD, USA). It uses signal amplification in cervical cellular samples to detect the five most frequent low-risk oncogenic HPV types (probe A: 6, 11, 42, 43, 44) and the 13 most frequent high-risk oncogenic HPV types (probe B: 16, 18, 31, 33, 35, 39, 51, 52, 53, 56, 58, 59, 68). It combines hybridisation of low-risk or high-risk oncogenic HPV RNA probes and the patient’s HPV DNA with an enzyme-linked immunosorbent assay (ELISA) (Figure 4). At a cut-off level of 1 pg/mL, it has the same sensitivity as the PCR test. At present, one technician can carry out 100 assays per day. In our opinion, the use of probe A has little, if any, clinical value because it detects benign, non-neoplastic HPV infections. By contrast, probe B identifies precancerous lesions that do have clinical significance. Many independent clinical studies have shown that HPV DNA testing (with probe B) improves significantly the detection of high-grade squamous intraepithelial lesions in women with borderline cytological abnormalities
The currently available scientific data show that the new techniques are likely to improve detection of cervical cancer and its precursors. The incidence of late-stage precancer (high-grade squamous intraepithelial lesions) and earlystage invasive disease peaks at age 35 to 45 years. The greatly improved sensitivity of the double test (HPV/Pap) should therefore lead to the detection of most of the clinically important lesions in this cohort of women. Removal of these lesions should avert many future cases of invasive disease. In the case of early invasive disease, detection at an early stage should confer better survival rates than those at more advanced clinical stages. Furthermore, HPV DNA testing has a unique advantage over cytology – its predictive value for the subsequent development of cervical cancer precursors in HPV-positive, but cytology-negative women.11 Women with persistent high-risk HPV types and with negative smears have a risk of developing cervical neoplasia at 2 to 4 years up to 100-fold greater than those with low-risk HPV types.11 The negative predictive value of double testing (HPV/Pap), which is of the order of 100% for high-grade
THE LANCET Oncology Vol 2 January 2001
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Review
Modern cervical cancer screening
studies have shown that the protection afforded by cytological screening begins to wane only after 3 years.1 In one study, high-grade squamous intraepithelial lesions developed at 48 months in only 0.06% of HPV-negative women compared with 7% of those who were HPV positive.10 Both the increased screening intervals, resulting in fewer repeat tests, and lower referral rates of women to gynaecologists and colposcopists are likely to improve compliance with new screening programmes. Another feature of HPV DNA testing is that it is suitable for self-sampling. In one experience with 1365 South African women aged 35 – 65 years, self-sampling for HPV testing (66.1%) detected as many cases of cervical cancer precursors as conventional cytology by health-care providers (67.9%).13 Self-testing for HPV DNA is particularly attractive in populations with social, cultural, and religious limitations on the acceptability of vaginal examinations.
Problems and solutions
Figure 4. Hybrid capture technology for HPV DNA testing. The patient’s HPV DNA (single-stranded after denaturation, shown in red) is combined with a mixture of 13 highly oncogenic RNA types (probe B, shown in purple). The hybrid is then captured by antibodies to HPV RNA (yellow). The complex is exposed to enzymatic digestion (green). The reaction results in the emission of light generated by a chemiluminescent substrate conjugated to the enzyme (blue). The light emission is translated into presence of HPV DNA, and the intensity of emitted light into viral load.
squamous intraepithelial lesions, is also very useful.9,10 This high rate should improve participation in new screening programmes, because people attending these programmes are obviously keen to know that they have no disease rather than that they have disease. The very high negative predictive value of the HPV/Pap test should permit longer intervals between screening tests. With respect to interval cancers, several studies have shown lack of progression of carcinoma in situ in the absence of high-risk HPV types.11 The fact that HPV-negative cervical cancers are almost never found is also reassuring.12 Furthermore, cervical carcinoma takes about 13 years to develop from normal cytology, and only about 40% of high-grade squamous intraepithelial lesions progress to clinical stage I cervical carcinoma in 8 – 12 years.11 As a result, the risk of developing cervical carcinoma during a 3 – 4 year interval in a woman with no history of abnormal cervical cytology before a negative double test at age 35 years must be extremely small. The rare undetected incident highgrade squamous intraepithelial lesion is very unlikely to develop into invasive cancer in less than 3 years. Several 30
Up to now, the rationale for developing new techniques was to provide better diagnostic accuracy which in turn might have the potential to modify our current policies with respect to cervical cancer screening programmes. In the short term, there will continue to be improvements in the new techniques to achieve their maximum clinical usefulness. Also, prospective data are forthcoming to help identify the most adequate techniques (and their combinations) and the most appropriate testing intervals to be used in screening programmes. The latency period of cervical cancer from the initial HPV infection is of the order of several decades.1 Therefore, the recent technological advances cannot be expected to affect mortality rates. However, from the results so far obtained, this goal will eventually be achieved. The social and medical costs of identifying HPVpositive, cytology-negative women must be considered, because such information could result in marital or family problems or difficulties with clinical management. Indeed, identification of these ‘HPV DNA carriers’ will become problematic if timing of HPV testing and the clinical significance of the HPV latency period are not handled appropriately. As has been suggested previously, these difficulties can be kept to a minimum if only women aged 35 and older undergo HPV DNA testing in a primary screening capacity. In this age-group, the prevalence of latent HPV infection in women with normal cytology is low (about 5%).9 By contrast, in younger women, particularly adolescents, the proportion of HPV-positive individuals is very high (20 – 40%), but the infection is transient in most, and they revert to normal via their cell-mediated immune system.10 In these women, HPV testing therefore has little or no clinical value. Restriction of HPV testing to women of 35 years and older prevents unnecessary colposcopy, cost, and anxiety. From a psychosocial point of view, most patients will understand and accept an explanation that the virus has long latency periods and is likely to have been contracted in the adolescent years. Finally, a woman found to be an HPV DNA carrier at age 35 years or older does not necessarily THE LANCET Oncology Vol 2 January 2001
For personal use only. Not to be reproduced without permission of The Lancet.
Modern cervical cancer screening
Review
have a false-positive result; the finding indicates that she is at risk of Start screening at developing cervical neoplasia from 20 years of age persistent HPV infection. National and international consensus guidelines should be TLC After 3 annual, negative tests, go to triennial TLC developed for the management of women aged 35 years and older who have positive test results for high-risk Triennial TLC HPV types but negative cytology. These women should probably be Negative Screen up to Reflex HPV testing 35 years of age examined by colposcopy; if found to 70 years of age (HPV/Pap) be free of disease, they should be followed up by cytology every 6 months for 2 years, as is currently Positive (either/both) recommended for minor-grade or equivocal abnormalities. The other Colposcopy option is to undertake repeat double testing at 12 months after the initial positive HPV test, to establish Figure 5. Proposed flow chart for primary cervical cancer screening with the new techniques. In HPV persistence and possible disease women with a negative HPV/Pap test at age 35 years, all subsequent Pap tests, preferably by thindevelopment. We reiterate that layer cytology (TLC) may be taken with intervals of 3 years or longer, provided that all samples are negative by cervical cytology. women with persistent, latent HPV infection are at high risk of developing radiotherapies by 47%, and was well below the acceptable cervical neoplasia.11 Women whose cytology remains health-care cost-effectiveness threshold of US$50 000 per negative or whose repeat HPV test result is normal life-year saved. can return to the routine cytological follow-up pool Other large-scale prospective studies in the USA (Figure 5). (the ASCUS, low-grade squamous epithelial lesion triage The expense of the new screening techniques will system [ALTS] trial) and in Europe, involving HPV DNA undoubtedly cause an increase in initial expenditure. testing, liquid-based and automated cytology in both However, large clinical trials in women with borderline diagnostic and primary screening modes are either atypia have shown that HPV DNA testing improves the underway or near completion. Several of these studies are efficacy of the secondary screening scheme by detecting using histological verification of the cervix of low-risk and more than 90% of high-grade cancer precursors which, after high-risk women who tested negative with the new screening all, are found in only a small subset (less than 10%) of methods. These studies will further our knowledge on the women with borderline or ASCUS cytology. At the same diagnostic performance, including specificity, of the new time, the HPV-based triage approach has the potential to techniques. Also, research is needed to assess the effect avoid unnecessary colposcopy in about 50% of women.8 of cervical cancer, its precursors, and their treatment on The cost of treating a detected high-grade squamous quality of life. intraepithelial lesion is much less than that incurred for treatment of invasive disease that has developed from a Conclusions It is only a matter of time before one of the last bastions of cytologically ‘missed’ lesion.14 The recent report from the Agency for Health Care morphology – manual cytology – will give way to new Policy and Research is the most comprehensive study so methods. Indeed, we are witnessing a major shift in disease far on the cost-effectiveness of cervical carcinoma detection and prognosis. Automated, robotic, and screening.1 It was the first study in which a 51% (as opposed molecular-based techniques are taking the forefront in to the traditional 80%) sensitivity estimate for the detecting cervical cancer and its precursors and will become conventional Pap test was recognised and used. Also, it an essential and indispensable part of modern screening modelled the improved medical outcome that can result programmes. These will use liquid-based thin-layer from improved sensitivity of the Pap test for various cytology, computer-assisted automated screening devices, screening intervals. The latter included cost per life-years and HPV DNA testing.3 The ultimate goal is to establish saved; cost per death from cervical carcinoma prevented; longer and safe screening intervals.1 The money saved by cost per case of cervical carcinoma prevented, and the increasing screening intervals should be spent on further number of morbid therapies avoided. The report assumed a clinical research on this topic and on urgently needed public 60% improvement of conventional Pap smear sensitivity awareness campaigns about cervical cancer. Let us not forget from use of thin-layer, liquid-based cytology and the that 471 000 women are diagnosed with invasive cervical generally accepted value of US$50 000 per life-year saved. carcinoma each year worldwide; about a third in the more The new method reduced the number of cases and deaths developed countries and two-thirds in less developed from cervical carcinoma by 51% with a 3-year screening countries die within 3 years of diagnosis of this otherwise interval, decreased the number of hysterectomies and preventable disease.15 THE LANCET Oncology Vol 2 January 2001
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Review Search strategy and selection criteria Evidence-based information on conventional and new technologies used for primary and secondary screening for cervix cancer and its precursors were included. Only large, clinical trials involving more than 1000 women, published in English after 1990 were included.
References
1 Evidence Report/Technology Abstract: number 5, Evaluation of Cervical Cytology (AHCPR Publication No 00-E010), Rockville, MD. Internet citation: www.ahcpr.gov/clinic/index.html1#evidence 2 Fahey MT, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. Am J Epidemiol 1995; 141: 680–89. 3 Austin RM, Ramzey I. Increased detection of epithelial cell abnormalities by liquid-based gynecologic cytology preparations: a review of accumulated data. Acta Cytol 1998; 42: 178–84. 4 Bishop JW, Bigner SH, Colgan TJ, et al. Multicenter masked evaluation of AutoCyte PREP thin layers with matched conventional smear including initial biopsy results. Acta Cytol 1998; 42: 189–97. 5 Diaz-Rosario LA, Kabawat SE. Performance of a fluid-based, thinlayer Papanicolaou smear method in the clinical setting of an independent laboratory and an outpatient screening population in New England. Arch Pathol Lab Med 1999; 123: 817–21.
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6 Vassilakos P, Saurel J, Rondez R. Direct-to-vial use of the AutoCyte PREP liquid-based preparation for cervical-vaginal specimens in three European Laboratories. Acta Cytol 1999; 43: 65–68. 7 Wilbur DC, Prey MU, Miller WM, Pawlick GF, Colgan TJ. The AutoPap System for primary screening in cervical cytology; comparing results of a prospective, intended-use study with routine manual practice. Acta Cytol 1998; 42: 214–20. 8 Manos MM, Kinney WK, Hurley LB, et al. Identifying women with cervical neoplasia using human papillomavirus DNA testing for equivocal Papanicolaou results. JAMA 1999; 281: 1605–10. 9 Cuzick J, Beverley E, Ho L, et al. HPV testing in primary screening of older women. Br J Cancer 1999; 81: 554–58. 10 Schiffman M, Herrero R, Hildesheim A, et al. HPV DNA testing in cervical cancer screening. JAMA 2000; 283: 87–93. 11 Rozendaal L, Walboomers JM, van der Linden JC, et al. PCR-based high-risk HPV test in cervical cancer screening gives objective assessment of women with cytomorphologically normal cervical smears. Int J Cancer 1996; 68: 766–69. 12 Walboomers JM, Jacobs MV, Manos M, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189: 12–19. 13 Wright TC, Denny L, Kuhn L, Pollack A, Lorincz A. HPV DNA testing of self-collected vaginal samples compared with cytologic screening to detect cervical cancer. JAMA 2000; 283: 81–86. 14 Lytwyn A, Gafni A, Sellors JW, et al. Economic evaluation of hybrid capture human papillomavirus testing in women with low-grade Papanicolaou smear abnormalities. J Lower Genit Tract Dis 1998; 2: 213–20. 15 Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999; 80: 827–41.
THE LANCET Oncology Vol 2 January 2001
For personal use only. Not to be reproduced without permission of The Lancet.
Modern cervical cancer screening
Cervical-cancer screening beyond the year 2000 Alex Ferenczy and Eduardo Franco
Evidence-based studies have shown that new techniques for cervical cancer screening have a higher diagnostic yield than conventional cervical cytology (Pap test). Automated screening devices that use liquid-based, thin-layer cytology and human papillomavirus DNA testing are likely to become the standard for routine primary screening for cervical cancer and its precursors in the 21st century. The increased initial expense of the new techniques will most certainly be absorbed by instituting longer intervals for safe primary screening, in both low-risk and high-risk populations. To make modern screening programmes even more effective, we must promote extensive public awareness campaigns about cervical cancer, a preventable disease. Lancet Oncol 2001; 2: 27–32
Despite the undeniable benefit from screening for cervical cancer with the Papanicolaou (Pap test), there is growing evidence of its limitations in terms of overall diagnostic performance. An extensive meta-analysis on the diagnostic accuracy of cervical cytology screening tests by the US Agency for Health Care Policy and Research1 found that only 86 of a total of 939 studies provided meaningful information. These studies used diagnostic standards such as histology or colposcopy and reported data on both sensitivity and specificity. Even more surprising was the finding that of the 86 studies, only three were not affected by verification bias. In these three studies, published between 1992 and 1995, disease was verified by the diagnostic gold standard, histology, in more than 2000 cytology-negative patients. The sensitivity of conventional cytology ranged from 29% to 56% (mean 49%) and specificity from 97% to 100%.1 The rates are similar to those in the meta-analysis by Fahey and colleagues (42% false-negative cytology).2 Falsenegative results have major medical, financial, and, in some parts of the world, legal implications. On the other hand, false-positive results lead to misuse of a substantial amount of health-care funds and generate unnecessary anxiety in women. The two major causes of false-negative cytology are sampling errors – about two thirds of cases. The remaining third are detection errors. False-positive results are mostly due to interpretation errors. In response to the pitfalls of manual cervical cytology, there has been development of high-technology devices with the potential to optimise Pap test screening programmes by reducing the false-negative and false-positive rates. THE LANCET Oncology Vol 2 January 2001
Figure 1. Thin-layer, liquid-based cytology. With this new technique, virtually all the cells collected from the cervix are suspended in the collection vial.
Technology overview Thin-layer, liquid-based cytology
The basic principle and advantage of this technique over the conventional Pap test is that the sample removed from the cervix is not placed on a glass slide but is rinsed from the sampler into a vial (Figure 1), which contains a cellAF is professor of Pathology and Obstetrics and Gynecology, McGill University and the Sir Mortimer B Davis-Jewish General Hospital, Montréal, Québec, Canada. EF is director of the Division of Epidemiology, Department of Oncology, at McGill University. Correspondence: Professor Alex Ferenczy, Department of Pathology, SMBD-Jewish General Hospital, 3755 Côte Ste Catherine Road, Montréal, Québec, Canada H3T 1E2. Tel: + 1 514 340 7526. Fax: +1 514 340 7542. Email: [email protected]
27
For personal use only. Not to be reproduced without permission of The Lancet.
Review
Modern cervical cancer screening
On the basis of those findings, the US Food and Drug Administration (FDA) approved the ThinPrep Pap test (1996) and AutoCyte PREP System (1999), respectively, as significantly superior and equivalent to the conventional Pap smear for the detection of precancerous and cancerous lesions of the cervix. By September 1999, the performance of the ThinPrep Pap test had been evaluated further in 154 872 lowrisk and high-risk women. In these studies, the cells collected from the cervix were placed in the collection vial (direct-to-vial protocol).5 All but one study used histological verification of positive Pap tests. Spectacular increases in the detection of precancerous lesions were observed, in comparison with historical controls tested with conventional Pap smears a year earlier. For example, the rate of detection of high-grade squamous intraepithelial lesions increased by 26% to 233% and that for low-grade lesions ranged from 40% to 331%. In the largest single study, on 59 539 women, there was a 39% decrease in the ratio of borderline or atypical squamous cells of undetermined significance (ASCUS) to low-grade squamous intraepithelial Figure 2. Cervical cytology. (a) Machine-processed thin-layer cervical sample (ThinPrep Pap), which is devoid of obscuring inflammatory and red blood cells, so interpretation is easier and more lesions.5 Most patients with borderline accurate. (b) Conventional Pap smear containing dense, inflammatory exudate that precludes Pap tests in fact have no disease. Any accurate interpretation of possible diagnostic cells. technique that can lower the preserving transport fluid, thus permitting the collection proportion of false-positive findings is most welcome, of virtually all cellular material in an optimum state. In because it has the potential to decrease substantially the the conventional Pap test, at best only 20% of the cells number of unnecessary colposcopic examinations and the are smeared onto the glass slide and the remaining 80% incremental costs for cervical biopsies and endocervical are lost with the discarded collection device. In the curettage. In the same study, the researchers showed a laboratory, the suspended cells are processed by robotic 99.8% reduction in rates of obscuring blood on slides, a preparation devices such as the ThinPrep (CYTYC Corp, 94.3% reduction in rates of obscuring inflammation, and MA, USA) or AutoCyte PREP System (TriPath Imaging, Inc, a 99.3% decrease in cases of poor collection. These findings NC, USA). Obscuring blood and inflammatory cells are are important contributors to better detection of abnormal automatically removed from the sample, which is then cells. In one multicentre European study, with manually transferred by machine in a thin layer onto a 13 mm prepared AutoCyte PREP direct-to-vial samples, there was (AutoCyte PREP) or 22 mm (ThinPrep) circular area of the an 89% reduction in the proportion of cytological slide (Figure 2). preparations that were “unsatisfactory” and a 74% reduction Large-scale prospective studies evaluated the clinical in the “satisfactory but limited by” category, a 79% performance of the ThinPrep Pap test compared with a improvement in the detection of high-grade squamous histological reference standard.1,3 They found sensitivities intraepithelial lesions, and a 43% decrease in the ASCUS of between 0.73 and 0.94 and specificities of 0.58 to 0.76.1 category compared with 88 569 contemporaneously collected The results of masked, multicentre clinical trials involving conventional Pap smears.6 Another key advantage of liquidmore than 16 000 cases (ThinPrep) and 8807 cases based cytology is that the residual cell suspension can be (AutoCyte PREP) of matched ‘split samples’ (ie Pap smear prepared for histology in cytologically equivocal cases, and first, thin-layer Pap test second) showed a significant most importantly, it can be used for panel testing. With the improvement in slide quality and an overall 18% higher ThinPrep system, one of the leading panel tests is related detection rate (ThinPrep) with the thin-layer approach than to detection of human papillomavirus (HPV) and the other with conventional cytology. AutoCyte PREP yielded to detection of Chlamydia trachomatis. Both tests have detection rates similar to the conventional Pap smear.4 been approved by the FDA. 28
THE LANCET Oncology Vol 2 January 2001
For personal use only. Not to be reproduced without permission of The Lancet.
Review
Modern cervical cancer screening
Computer-assisted automated cytology
At present, the only automated scanning system approved by the FDA for quality control and primary screening purposes is the AutoPap screening system (TriPath Imaging). It scans about 200 conventional Pap smears a day by obtaining images with a high-resolution, high-speed video microscope. The images are digitised, and the morphometric algorithms identify and classify both individual cells and the Pap smear as a whole. Automated scanning of manually screened smears with a negative proportion of 10% as part of quality assurance outperformed human review by a factor of between five and seven, and in primary screening mode Figure 3. Computer-assisted classification of abnormal cells. The left shoulder of the bell-shaped (ie for women without symptoms), it curve represents the 25% least abnormal smears or the ‘sort rate’. These smears do not need detected 100% and 96% of the human review because they are negative. The rest of the smears have cellular abnormalities and need to be reviewed manually. Those located in the right shoulder of the curve are hierarchically the invasive cancers and high-grade most abnormal and need to be reviewed by a supervisor in a quality control mode. squamous intraepithelial lesions, respectively.7 In May 1998, the FDA approved the device at a (secondary screening).8 Similarly, in nine independent 25% ‘sort rate’ in low-risk (not attending a hospital clinic) population-based, international, primary screening studies populations of women. In other words, the device identifies on more than 30 000 women, HC-II was uniformly more the 25% of smears that do not need human review because sensitive for detecting cervical cancers and high-grade they are definitely negative. Conversely, the 75% of the precursors (85% – 100% compared with the 40% – 78% rate smears more likely to be abnormal must be manually for conventional cervical cytology). Combination of the screened by the cytotechnologist (Figure 3). Elsewhere, results of cytology and HPV testing leads to 100% detection including Canada, Europe, and Asia, the device is used at a of high-grade squamous intraepithelial lesions.9,10 Rates of sort rate of 50%. The reason for the differential sort rate referral to colposcopy were 12.5% and 7.0% as a result of approval is that the manufacturer’s submission for approval positive HPV DNA tests and conventional cytology, in the USA preceded the submissions in other countries by respectively. In a population-based primary screening study about a year; by the time of those submissions, further on 2988 women aged 35 years or older, the rate of Papfavourable data had accrued on the diagnostic accuracy of negative HPV-positive women (HPV DNA carriers) was only 4.8% at the recommended cut-off of 1 pg/mL.9 the AutoPap screening device. HPV DNA testing
Prospects
So far, the only HPV test approved by the FDA for commercial clinical use is the Hybrid Capture-II microtitre assay (HC-II/Digene Corp, MD, USA). It uses signal amplification in cervical cellular samples to detect the five most frequent low-risk oncogenic HPV types (probe A: 6, 11, 42, 43, 44) and the 13 most frequent high-risk oncogenic HPV types (probe B: 16, 18, 31, 33, 35, 39, 51, 52, 53, 56, 58, 59, 68). It combines hybridisation of low-risk or high-risk oncogenic HPV RNA probes and the patient’s HPV DNA with an enzyme-linked immunosorbent assay (ELISA) (Figure 4). At a cut-off level of 1 pg/mL, it has the same sensitivity as the PCR test. At present, one technician can carry out 100 assays per day. In our opinion, the use of probe A has little, if any, clinical value because it detects benign, non-neoplastic HPV infections. By contrast, probe B identifies precancerous lesions that do have clinical significance. Many independent clinical studies have shown that HPV DNA testing (with probe B) improves significantly the detection of high-grade squamous intraepithelial lesions in women with borderline cytological abnormalities
The currently available scientific data show that the new techniques are likely to improve detection of cervical cancer and its precursors. The incidence of late-stage precancer (high-grade squamous intraepithelial lesions) and earlystage invasive disease peaks at age 35 to 45 years. The greatly improved sensitivity of the double test (HPV/Pap) should therefore lead to the detection of most of the clinically important lesions in this cohort of women. Removal of these lesions should avert many future cases of invasive disease. In the case of early invasive disease, detection at an early stage should confer better survival rates than those at more advanced clinical stages. Furthermore, HPV DNA testing has a unique advantage over cytology – its predictive value for the subsequent development of cervical cancer precursors in HPV-positive, but cytology-negative women.11 Women with persistent high-risk HPV types and with negative smears have a risk of developing cervical neoplasia at 2 to 4 years up to 100-fold greater than those with low-risk HPV types.11 The negative predictive value of double testing (HPV/Pap), which is of the order of 100% for high-grade
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studies have shown that the protection afforded by cytological screening begins to wane only after 3 years.1 In one study, high-grade squamous intraepithelial lesions developed at 48 months in only 0.06% of HPV-negative women compared with 7% of those who were HPV positive.10 Both the increased screening intervals, resulting in fewer repeat tests, and lower referral rates of women to gynaecologists and colposcopists are likely to improve compliance with new screening programmes. Another feature of HPV DNA testing is that it is suitable for self-sampling. In one experience with 1365 South African women aged 35 – 65 years, self-sampling for HPV testing (66.1%) detected as many cases of cervical cancer precursors as conventional cytology by health-care providers (67.9%).13 Self-testing for HPV DNA is particularly attractive in populations with social, cultural, and religious limitations on the acceptability of vaginal examinations.
Problems and solutions
Figure 4. Hybrid capture technology for HPV DNA testing. The patient’s HPV DNA (single-stranded after denaturation, shown in red) is combined with a mixture of 13 highly oncogenic RNA types (probe B, shown in purple). The hybrid is then captured by antibodies to HPV RNA (yellow). The complex is exposed to enzymatic digestion (green). The reaction results in the emission of light generated by a chemiluminescent substrate conjugated to the enzyme (blue). The light emission is translated into presence of HPV DNA, and the intensity of emitted light into viral load.
squamous intraepithelial lesions, is also very useful.9,10 This high rate should improve participation in new screening programmes, because people attending these programmes are obviously keen to know that they have no disease rather than that they have disease. The very high negative predictive value of the HPV/Pap test should permit longer intervals between screening tests. With respect to interval cancers, several studies have shown lack of progression of carcinoma in situ in the absence of high-risk HPV types.11 The fact that HPV-negative cervical cancers are almost never found is also reassuring.12 Furthermore, cervical carcinoma takes about 13 years to develop from normal cytology, and only about 40% of high-grade squamous intraepithelial lesions progress to clinical stage I cervical carcinoma in 8 – 12 years.11 As a result, the risk of developing cervical carcinoma during a 3 – 4 year interval in a woman with no history of abnormal cervical cytology before a negative double test at age 35 years must be extremely small. The rare undetected incident highgrade squamous intraepithelial lesion is very unlikely to develop into invasive cancer in less than 3 years. Several 30
Up to now, the rationale for developing new techniques was to provide better diagnostic accuracy which in turn might have the potential to modify our current policies with respect to cervical cancer screening programmes. In the short term, there will continue to be improvements in the new techniques to achieve their maximum clinical usefulness. Also, prospective data are forthcoming to help identify the most adequate techniques (and their combinations) and the most appropriate testing intervals to be used in screening programmes. The latency period of cervical cancer from the initial HPV infection is of the order of several decades.1 Therefore, the recent technological advances cannot be expected to affect mortality rates. However, from the results so far obtained, this goal will eventually be achieved. The social and medical costs of identifying HPVpositive, cytology-negative women must be considered, because such information could result in marital or family problems or difficulties with clinical management. Indeed, identification of these ‘HPV DNA carriers’ will become problematic if timing of HPV testing and the clinical significance of the HPV latency period are not handled appropriately. As has been suggested previously, these difficulties can be kept to a minimum if only women aged 35 and older undergo HPV DNA testing in a primary screening capacity. In this age-group, the prevalence of latent HPV infection in women with normal cytology is low (about 5%).9 By contrast, in younger women, particularly adolescents, the proportion of HPV-positive individuals is very high (20 – 40%), but the infection is transient in most, and they revert to normal via their cell-mediated immune system.10 In these women, HPV testing therefore has little or no clinical value. Restriction of HPV testing to women of 35 years and older prevents unnecessary colposcopy, cost, and anxiety. From a psychosocial point of view, most patients will understand and accept an explanation that the virus has long latency periods and is likely to have been contracted in the adolescent years. Finally, a woman found to be an HPV DNA carrier at age 35 years or older does not necessarily THE LANCET Oncology Vol 2 January 2001
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Modern cervical cancer screening
Review
have a false-positive result; the finding indicates that she is at risk of Start screening at developing cervical neoplasia from 20 years of age persistent HPV infection. National and international consensus guidelines should be TLC After 3 annual, negative tests, go to triennial TLC developed for the management of women aged 35 years and older who have positive test results for high-risk Triennial TLC HPV types but negative cytology. These women should probably be Negative Screen up to Reflex HPV testing 35 years of age examined by colposcopy; if found to 70 years of age (HPV/Pap) be free of disease, they should be followed up by cytology every 6 months for 2 years, as is currently Positive (either/both) recommended for minor-grade or equivocal abnormalities. The other Colposcopy option is to undertake repeat double testing at 12 months after the initial positive HPV test, to establish Figure 5. Proposed flow chart for primary cervical cancer screening with the new techniques. In HPV persistence and possible disease women with a negative HPV/Pap test at age 35 years, all subsequent Pap tests, preferably by thindevelopment. We reiterate that layer cytology (TLC) may be taken with intervals of 3 years or longer, provided that all samples are negative by cervical cytology. women with persistent, latent HPV infection are at high risk of developing radiotherapies by 47%, and was well below the acceptable cervical neoplasia.11 Women whose cytology remains health-care cost-effectiveness threshold of US$50 000 per negative or whose repeat HPV test result is normal life-year saved. can return to the routine cytological follow-up pool Other large-scale prospective studies in the USA (Figure 5). (the ASCUS, low-grade squamous epithelial lesion triage The expense of the new screening techniques will system [ALTS] trial) and in Europe, involving HPV DNA undoubtedly cause an increase in initial expenditure. testing, liquid-based and automated cytology in both However, large clinical trials in women with borderline diagnostic and primary screening modes are either atypia have shown that HPV DNA testing improves the underway or near completion. Several of these studies are efficacy of the secondary screening scheme by detecting using histological verification of the cervix of low-risk and more than 90% of high-grade cancer precursors which, after high-risk women who tested negative with the new screening all, are found in only a small subset (less than 10%) of methods. These studies will further our knowledge on the women with borderline or ASCUS cytology. At the same diagnostic performance, including specificity, of the new time, the HPV-based triage approach has the potential to techniques. Also, research is needed to assess the effect avoid unnecessary colposcopy in about 50% of women.8 of cervical cancer, its precursors, and their treatment on The cost of treating a detected high-grade squamous quality of life. intraepithelial lesion is much less than that incurred for treatment of invasive disease that has developed from a Conclusions It is only a matter of time before one of the last bastions of cytologically ‘missed’ lesion.14 The recent report from the Agency for Health Care morphology – manual cytology – will give way to new Policy and Research is the most comprehensive study so methods. Indeed, we are witnessing a major shift in disease far on the cost-effectiveness of cervical carcinoma detection and prognosis. Automated, robotic, and screening.1 It was the first study in which a 51% (as opposed molecular-based techniques are taking the forefront in to the traditional 80%) sensitivity estimate for the detecting cervical cancer and its precursors and will become conventional Pap test was recognised and used. Also, it an essential and indispensable part of modern screening modelled the improved medical outcome that can result programmes. These will use liquid-based thin-layer from improved sensitivity of the Pap test for various cytology, computer-assisted automated screening devices, screening intervals. The latter included cost per life-years and HPV DNA testing.3 The ultimate goal is to establish saved; cost per death from cervical carcinoma prevented; longer and safe screening intervals.1 The money saved by cost per case of cervical carcinoma prevented, and the increasing screening intervals should be spent on further number of morbid therapies avoided. The report assumed a clinical research on this topic and on urgently needed public 60% improvement of conventional Pap smear sensitivity awareness campaigns about cervical cancer. Let us not forget from use of thin-layer, liquid-based cytology and the that 471 000 women are diagnosed with invasive cervical generally accepted value of US$50 000 per life-year saved. carcinoma each year worldwide; about a third in the more The new method reduced the number of cases and deaths developed countries and two-thirds in less developed from cervical carcinoma by 51% with a 3-year screening countries die within 3 years of diagnosis of this otherwise interval, decreased the number of hysterectomies and preventable disease.15 THE LANCET Oncology Vol 2 January 2001
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Review Search strategy and selection criteria Evidence-based information on conventional and new technologies used for primary and secondary screening for cervix cancer and its precursors were included. Only large, clinical trials involving more than 1000 women, published in English after 1990 were included.
References
1 Evidence Report/Technology Abstract: number 5, Evaluation of Cervical Cytology (AHCPR Publication No 00-E010), Rockville, MD. Internet citation: www.ahcpr.gov/clinic/index.html1#evidence 2 Fahey MT, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. Am J Epidemiol 1995; 141: 680–89. 3 Austin RM, Ramzey I. Increased detection of epithelial cell abnormalities by liquid-based gynecologic cytology preparations: a review of accumulated data. Acta Cytol 1998; 42: 178–84. 4 Bishop JW, Bigner SH, Colgan TJ, et al. Multicenter masked evaluation of AutoCyte PREP thin layers with matched conventional smear including initial biopsy results. Acta Cytol 1998; 42: 189–97. 5 Diaz-Rosario LA, Kabawat SE. Performance of a fluid-based, thinlayer Papanicolaou smear method in the clinical setting of an independent laboratory and an outpatient screening population in New England. Arch Pathol Lab Med 1999; 123: 817–21.
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6 Vassilakos P, Saurel J, Rondez R. Direct-to-vial use of the AutoCyte PREP liquid-based preparation for cervical-vaginal specimens in three European Laboratories. Acta Cytol 1999; 43: 65–68. 7 Wilbur DC, Prey MU, Miller WM, Pawlick GF, Colgan TJ. The AutoPap System for primary screening in cervical cytology; comparing results of a prospective, intended-use study with routine manual practice. Acta Cytol 1998; 42: 214–20. 8 Manos MM, Kinney WK, Hurley LB, et al. Identifying women with cervical neoplasia using human papillomavirus DNA testing for equivocal Papanicolaou results. JAMA 1999; 281: 1605–10. 9 Cuzick J, Beverley E, Ho L, et al. HPV testing in primary screening of older women. Br J Cancer 1999; 81: 554–58. 10 Schiffman M, Herrero R, Hildesheim A, et al. HPV DNA testing in cervical cancer screening. JAMA 2000; 283: 87–93. 11 Rozendaal L, Walboomers JM, van der Linden JC, et al. PCR-based high-risk HPV test in cervical cancer screening gives objective assessment of women with cytomorphologically normal cervical smears. Int J Cancer 1996; 68: 766–69. 12 Walboomers JM, Jacobs MV, Manos M, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189: 12–19. 13 Wright TC, Denny L, Kuhn L, Pollack A, Lorincz A. HPV DNA testing of self-collected vaginal samples compared with cytologic screening to detect cervical cancer. JAMA 2000; 283: 81–86. 14 Lytwyn A, Gafni A, Sellors JW, et al. Economic evaluation of hybrid capture human papillomavirus testing in women with low-grade Papanicolaou smear abnormalities. J Lower Genit Tract Dis 1998; 2: 213–20. 15 Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999; 80: 827–41.
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