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Cytopathology of false negatives preceding cervical carcinoma
Cytopathology of false negatives preceding cervical carcinoma R i c h a r d M. D e M a y , M D
Chicago, Illinois The Papanicolaou smear has been a remarkably effective tool in cancer prevention, but it is not a perfect test.Although the most important factor in failure of cervical cancer prevention is lack of adequate screening, other factors include problems with sampling, interpretation, and effective clinical follow-up. A small number of rapidly developing cervical cancers probably also arise in the interval between Papanicolaou smear screenings. Consequently, cervical cancer will develop in some women despite appropriate screening. This article will analyze some of the problems relating to diagnostic errors, which include abnormal cells that are few (<100), small, or bland. (Am J Obstet Gynecol 1996;175:1110-3.)
Key words; False-negative Papanicolaou smear, cervical carcinoma
Although no better cancer prevention test has ever been devised, Papanicolaou smear interpretation has been shown in many studies to be associated with a significant false-negative rate, ranging from 0% 1up to 94% 2 in 13 published trials (Table I). No cytology laboratory, however well run, is completely i m m u n e to such errors. Cervical cytologic analysis c a r r i e s with it an irreducible diagnostic error rate of at least 5% to 10% (i.e., at least 1 in every 10 to 20 positive cases will be missed in routine screening)? Other problems are also associated with use of the Papanicolaou smear, such as sampling errors and, more important, failure of women to be adequately screened. This report describes and evaluates some of the causes of interpretation errors resulting in false-negative diagnoses in cervical cytologic analysis.
whom high-grade cervical dysplasia or cervical carcinoma subsequently was diagnosed. The interval between the last negative report and the diagnosis ranged from an average of 9.3 months 2 to as long as 19 years4; in most cases, intervals were 5 years or less. The proportion of s m e a r s reclassified from "normal" to "abnormal" r a n g e s from 0% ~ to 94%2; with only two exceptions, however, at least a quarter of the cases analyzed were reclassified. Of the total 655 reported smears, 340, or about half (51.9%), were reclassified as abnormal. The definition of abnormal, however, ranged from atypical squamous cells of u n d e t e r m i n e d significance to malignant cells. This large proportion of reclassified smears indicates an extremely high false-negative rate. False-negative studies
Rescreening "negative" cervical smears preceding a "positive" diagnosis False-negative cervical cytologic reports are significant for at least three reasons: first, and most important, they represent the possibility that serious or even life-threatening disease has gone undetected and therefore untreated; second, they can provide an opportunity to evaluate whether a "rapidly progressive" form of cervical cancer truly exists or whether cases so diagnosed are, in fact, related to prior false negatives; and third, a study of false negatives can reveal areas where e r r o r s are most likely to be made and where corrective action might be possible. Table I shows results from 13 published rescreening studies involving women with prior negative smears in From the Section of Cytopathology, University of Chicago. Reprint requests: Richard M. DeMay, MD, Professorof Clinical Pathology, Direct~ Section of Cytopathology, University of Chicago, MARP 212, MC 2050, 5841 S. Maryland Ave., Chicago, IL 60637. Copyright © 1996 by Mosby-Yea,rBook, Inc. 0002-9378/96 $5.00+ 0 6/0/75667 1110
Hatem and Wilbur (1995) reviewed 17 "negative" smears immediately preceding a Papanicolaou smear showing high-grade squamous intraepithelial lesions or cervical carcinoma. 2 The time interval between the negative and positive smears averaged 9.3 months. The authors found that only 1 (6%) of 17 smears represented true negatives and, hence, possible rapid progression. The remaining 16 (94%) of 17 smears were considered to be false negatives. In 12 (75%) of these 16 cases, the s m e a r s contained fewer than 100 abnormal cells, making detection difficult. Most of these patients (88%) had histories of abnormal Papanicolaou smears. The missed lesions were analyzed: 6% were reclassified as "atypical cells of u n d e t e r m i n e d type and origin," 31% as "atypical cells" or "slight dysplasia," 31% as "highgrade squamous intraepithelial lesions," and 69% as "atypical immature squamous metaplastic cells" (some cases carried multiple diagnoses). The atypical immature squamous cells were described as very small cells, with high nuclear/cytoplasmic ratios, hyperchromatic nuclei, and occasionally irregular nuclear membranes and were
Volume 175, Number 4, Part 2 AmJ Obstet Gynecol
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Table I. False-negative smears in cervical cytologic analysis "Negative" smears reclassified as "abnormal" Investigator (year)
''Negative" cytologic findings preceding "positive"
"'Negative" smears reviewed (n)
No.
%
Hatem and Wilbur (1995) 2 Attwood et al. (1985) 0 Robertson and Woodend (1993) 5 Rylander (1977) 10 Berkowitz et al. (1979) !~ Paterson et al. (1984) 12 Wain et al. (1992) 13 Sherman and Kelly (1992) 4 Kristensen et al. (1991) 14 Peters et al. (1988) 15 Walker et al. (1983) 16 Morell et al. (1982) 17 Liu (1967) 1
Averaged 9.3 mo Within 5 yr Up to 12 yr 4 to 5 )Jr Within 2 yr Within 10 yr Within 2 yr Median 93.5 mo Within 3 yr Within 3 yr Within 5 yr Within 3 yr 3.4yr
17 28 139 56 13 58 30 123 96 32 11 36 16 655
16 20 92 35 8 34 16 65 39 10 3 2 0 340
94.1 71.4 66.2 62.5 61.5 58.6 53.3 52.8 40.6 31.3 27.3 5.6 0 51.91
TOTAI~S
frequently f o u n d isolated in the smears. These cells resembled histiocytes or other b e n i g n cells. Harem a n d Wilbur 2 c o n c l u d e d that errors of screening or interpretation were i m p o r t a n t causes of false-negative reports in this p o p u l a t i o n a n d that these errors were related to the presence of few a b n o r m a l cells or small a b n o r m a l cells. O f clinical importance, however, is that patients with false-negative Papanicolaou smears usually have a history of a b n o r m a l smears. S h e r m a n a n d Kelly (1992) reviewed a total of 123 "negative" smears from 20 w o m e n in whom biopsyproved grade 3 cervical intraepithelial neoplasia (18 cases) or invasive cervical carcinoma (2 cases) developed, 4 The disease developed after three or m o r e "negative" Papanicolaou smears, with a m e d i a n interval of 93.5 m o n t h s (range 48 to 229 months). O n review, 23% of these smears were true negatives. A n o t h e r 25% of cases were reclassified as limited adequacy or completely unsatisfactory for evaluation (in 11% there was n o atypia, b u t the endocervical c o m p o n e n t was inadequate; 14% were unsatisfactory); 30% were reclassified as at)3oical squamous cells of u n d e t e r m i n e d significance; a n d 22% (28 smears) were considered false negatives, i n c l u d i n g 8% low-grade squamous intraepithelial lesions, 12% highgrade squamous intraepithelial lesions, a n d 2% squamous intraepithelial lesions of i n d e t e r m i n a t e degree. Analysis of these 28 false-negative smears showed that 14 (50%) c o n t a i n e d only a few (<100) cells diagnostic of squamous intraepithelial lesions. In five cases diagnostic cells were obscured by inflammation, a n d in four cases the a b n o r m a l cells were primarily present in tissue fragments or sheets that were difficult to distinguish from reactive or atrophic changes. Five false-negative smears involved small grade 3 cervical intraepithelial neoplasia cells with high nuclear/cytoplasmic ratios, irregular n u c l e a r outlines, a n d m i n i m a l hyperchromasia. These cells resembled cells of squamous metaplasia. Clinically, slightly more t h a n half the patients (55%)
had r e c u r r e n t Trichomonas infections. Approximately o n e third (35%) had unsatisfactory smears that had b e e n inappropriately reported as negative. O n e patient in four had a lapse in screening of 2 or more years. Interestingl); most patients (89%) had predominately endocervical lesions that were frequently located high in the endocervical canal. Even though an endocervical c o m p o n e n t was present in the smear, sampling of the lesion was still considered poor. Only 35% of patients had smears diagnostic of low-grade squamous intraepithelial lesions; all other a b n o r m a l smears were either atypical squamous cells of u n d e t e r m i n e d significance or high-grade squamous intraepithelial lesions. The authors c o n c l u d e d that most patients only appeared to have repeatedly negative Papanicolaou smears, when in reality most were either unsatisfactory for evaluation or extremely difficult to screen. 4 A long history of negative smears, especially in patients with multiple infections or heavy inflammation, should not, said the authors, be taken as proof of absence of disease. Many of the false negatives in this study were related to the presence of few diagnostic cells in the smear; most of these lesions were high in the endocervix a n d poorly sampled, despite the presence of an endocervical c o m p o n e n t . Problems in cytologic interpretation were also related to small, abnormal grade 3 cervical intraepithelial neoplasia cells that resembled squamous metaplasia. The description of these cells is similar to that of the cells in H a t e m a n d Wilbur's article s a n d characterizes cellular morphologic features that are particularly problematic for cervical cytologic screening. The differential diagnosis of tissue fragments can be also problematic in some cases; fragments derived from significant cervical lesions can be difficult to distinguish from b e n i g n processes such as atrophy or reactive changes. Finally the authors concluded that atypical squamous cells of u n d e t e r m i n e d significance may be the only identifiable cytolo~c abnormality d u r i n g the early d e v e l o p m e n t of high-grade squa-
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mous intraepithelial lesions. Because of false negatives, reports of rapidly progressing cervical cancers should be viewed cautiously. A third study addressed the issue of false negatives in cervical cytologic testing. 5 The investigators reviewed 139 negative smears from 102 women, o b t a i n e d up to 12 years before a diagnosis of cervical cancer. The diagnoses included squamous cell carcinoma (83) a n d adenocarcin o m a (20). Review showed that only 47 (34%) of these 139 smears were true negatives a n d that most of these (64%) were deficient with respect to the endocervical c o m p o n e n t . The r e m a i n i n g 92 smears (66%) were false negatives, i n c l u d i n g 74 squamous cell carcinomas a n d 18 adenocarcinomas. In analysis of the false negatives, more than a quarter of cases (26 of 92 smears, or 28%) were characterized by the presence of few a b n o r m a l cells; most of these cases (18 of 26, or 69%) had a n inadequate endocervical c o m p o n e n t ; a n d of the r e m a i n i n g false negatives 7 of 26 had i n f l a m m a t o r y or postmenopausal changes. A n o t h e r quarter of the smears (24 of 92) had a b n o r m a l ceils present mainly as tissue fragments, with few single cells. This finding occurred in cases of both squamous a n d glandular abnormalities. Nearly half of the cases (42 of 92) had m o d e r a t e n u m b e r s or many abnormal cells present o n review. The authors c o n c l u d e d that smears with few a b n o r m a l cells are unlikely to be detected in routine screening. 5 Tissue fragments, or"microbiopsies," may be associated with either squamous or glandular neoplasia, a n d screeners are n o t sufficiently aware of the diagnostic i m p o r t a n c e of these fragments. I n f l a m m a t o r y a n d postmenopausal changes make diagnosis more difficult. In addition, a n inadequate endocervical c o m p o n e n t is associated with false negatives. Almost half of these false-negative cases contained n u m e r o u s a b n o r m a l cells o n review, yet screeners still failed to detect the a b n o r m a l cells. An i m p o r t a n t cause of false negatives was d e e m e d to be the h u m a n factor: screener fatigue. The same research group studied 62 smears from 62 patients with cervical carcinoma. 6 They reviewed findings in the earliest smear originally diagnosed as showing any abnormality. The smears were taken u p to 18 years before the diagnosis of cervical cancer: 60 patients had sqnamous cell carcinoma; two patients had adenocarcinoma. T h e majority of these smears were j u d g e d to be false negatives b u t also i n c l u d e d true positives in patients lost to follow-up. O n review, 51 (82%) of the 62 smears showed definite severe dyskaryosis (the British term for dysplasia) or grade 3 cervical intraepithelial neoplasia. A n o t h e r 6, or 10%, showed probable severe dyskaryosis; in these cases, smears c o n t a i n e d few a b n o r m a l cells or indicated inflammation. The r e m a i n d e r showed moderate dyskaryosis (4) or h u m a n papillomavirus-associated changes (1). I n n o case was mild dyskaryosis (mild dysplasia) diagnosed in a patient.
October 1996 AmJ Obstet Gynecol
The authors concluded that mild dyskaryosis (i.e., mild dysplasia) is only rarely a f o r e r u n n e r of invasive carcinoma. 6 Therefore, cancer prevention d e p e n d s primarily o n the detection a n d t r e a t m e n t of severe dyskaryosis (severe dysplasia) or grade 3 cervical intraepithelial neoplasia. These findings would support newer concepts that cervical carcinoma generally arises from an aggressive grade 3 cervical intraepithelial neoplasia lesion, widely present in the cervix a n d established years before invasion, rather than from the progression of low-grade cervical intraepithelial neoplasia. T h e authors' findings support the Bethesda System n o m e n c l a t u r e of low- a n d highgrade squamous intraepithelial lesions. 7 The final report to be discussed addresses screening results in the n o r m a l screening e n v i r o n m e n t compared with a test screening environment. 8Archival material consisted of five "false-negative" smears (smears initially diagnosed as n o r m a l b u t later f o u n d to contain cancer cells) a n d five "u-ue-positive" (smears diagnosed as mal i g n a n t from patients with cancer). The "false-negative" smears were considered diagnostically difficult cases; the "true-positive" smears, diagnostically straightforward. The five prior false-negative smears were tested with five screeners u n d e r n o r m a l screening cofiditions, without the screener's knowledge (by adding the cases in a b l i n d e d fashion to the daily workload of routine screening). The five prior true positives were similarly tested with five screeners u n d e r n o r m a l screening conditions, without the screener's knowledge. T h e n the false negatives were separately tested, b u t this time u n d e r test conditions, that is, with the screeners b e i n g forewarned that the routine cases were seeded with a b n o r m a l Papanicolaou smears. U n d e r n o r m a l screening conditions (i.e., the screeners were unaware they were being tested), only o n e correct detection out of a possible 25 was made (5 false-negative slides times 5 screeners). Even this single detection was a fluke, because the screener actually f o u n d an incompletely removed m a r k i n g dot rather than the m a l i g n a n t cells themselves. In four additional cases, the cases were called a b n o r m a l b u t n o t cancer. All true-positive cases were correctly identified o n the first review. In the second part of the test, in which the screeners were aware that they were being tested, the detection rate was considerably better; cancer was detected in seven of eight prior false-negative smears. However, this high detection rate occurred only at the expense of a threefold rise in the n u m b e r of smears identified as "suspicious" or "positive" a n d a d o u b l i n g of screening times. O n analysis, the false-negative smears were f o u n d to differ from true-positive smears in several ways. The falsenegative smears c o n t a i n e d few or no large tissue fragments; there were few or n o single a b n o r m a l cells; a n d the a b n o r m a l cells were small, with small nuclei, little pleomorphism, a n d m i n i m a l hyperchromasia. The authors c o n c l u d e d that it is virtually impossible to avoid all
Volume i75, Number 4, Part 2 AmJ Obstet Gynecol
false negatives. 8 The tedium of screening leads to false negatives, b u t u n d e r conditions of extreme alertness, although the false-negative rate is diminished, it comes at the expense of increased false positives a n d screening times and, therefore, diminished laboratory productivity. Comment
The five articles discussed provide examples of the typical cytodiagnostic problems in routine screening that lead to false-negative reports. Chief a m o n g these are smears c o n t a i n i n g few a b n o r m a l cells: detection of abnormality in slides with fewer than 100 a b n o r m a l cells was markedly poorer than in slides with m a n y such ceils. It is possible that 100 a b n o r m a l cells represent a diagnostic threshold below which detection is unreliable. The problem becomes c o m p o u n d e d when the a b n o r m a l cells are small a n d therefore n o t only difficult to detect b u t difficult to interpret once detected. These small a b n o r m a l cells can resemble ordinary i m m a t u r e metaplastic ceils or even histiocytes. 3 The diagnostic d i l e m m a posed by tissue fragments, or "hyperchromatic crowded groups" has n o t b e e n sufficiently emphasized. 3 Hyperchromatic crowded groups are the general morphologic appearance of a variety of serious lesions in the Papanicolaou smear, including in situ or invasive carcinoma, b u t similar groups are also associated with b e n i g n entities, such as atrophy or e n d o m e t r i a l cells. The differential diagnosis of hyperchromatic crowded groups can be very difficult in some cases. Abnormality of glandular cells can be even more difficult to accurately identify a n d diagnose than squamous abnormalities. High-grade, small-cell lesions located high in the endocervical canal are particularly likely to evade detection. In addition, atypical squamous cells of u n d e t e r m i n e d significance are an i m p o r t a n t diagnosis a n d may be the only abnormality detectable in the Papanicolaou smear preceding the d e v e l o p m e n t of high-grade squamous intraepithelial lesions. Most patients with false-negative Papanicolaou smears have histories of a b n o r m a l smears, b u t multiple "negatives" do n o t completely exclude a clinically significant lesion. A n o t h e r i m p o r t a n t cause of app a r e n t false negatives is limited or unsatisfactory smears that are n o t properly identified as such. Finally, although rapid progression of cervical carcinoma may occur, m a n y cases represent prior false-negative diagnoses. In summary, false negatives are inevitable in routine cervical cytologic screening. Patients a n d clinicians alike should be w a r n e d that n o cytology laboratory is completely free of diagnostic errors; errors occur at a low b u t well-docmnented a n d probably irreducible rate of at least 5% to 10% in r o u t i n e screening. 3 Unfortunately; this means that cervical carcinoma will develop in some w o m e n despite appropriate screening. To reduce the risk of this tragic outcome, w o m e n should obtain regular Papanicolaou smears even if they have long histories of n o r m a l
DeMay 1113
cervical cytologic findings. All a b n o r m a l results, including a diagnosis of atypical squamous ceils of u n d e t e r m i n e d significance, should be followed up as clinically appropriate with, of course, biopsy of any visible suspicious lesions a n d investigation of any suspicious symptoms. Finally, it is i m p o r t a n t when j u d g i n g the Papanicolaou smear to realize that, although it is imperfect, n o other test has b e e n as successful in preventing a n d detecting cancer. W o m e n are more likely to have cervical cancer related to inadequate screening than to misdiagnosed Papanicolaou smears?
REFERENCES
1. Liu W. Positive smears in previously screened patients (certain cytologic findings of public health importance). Acta Cytol 1967;11:193-8. 2. Harem F, Wilbur D. High-grade cervical lesions following negative Papanicolaou smears: false-negative cervical cytology or rapid progression. Diagn Cytopathol 1995;12:13541. 3. DeMay RM. Failure of the Pap smear. In: The art & science of cytopathology. Chicago: ASCP Press, 1996:141-7. 4. Sherman ME, Kelly D. High-grade squamous intraepithelial lesions and invasive carcinoma following the report of three negative Papanicotaou smears: screening failures or rapid progression? Mod Pathol 1992;5:337-42. 5. RobertsonJH, Woodend B. Negative cytology preceding cervical cancer: causes and prevention..] Clin Pathot 1993;46: 700-2. 6. Robertson JH, Woodend B, Elliott H. Cytological changes preceding cervical cancer. J Clin Pathol 1994;47:278-9. 7. Sherman ME, Schiffman MH, Erozan YS, Wacholder S, Kurman RJ. The Bethesda System. A proposal for reporting abnormal cervical smears based on the reproducibility of cytopathologic diagnoses, Arch Pathol Lab Med 1992;116: 1155-8. 8. Bosch MMC, Petronella ElVl, Rietveld-Scheffers CT, Boon ME. Characteristics of false-negative smears tested in the normal screening situation. Acta Gytol 1992;36:711-6. 9. Attwood ME, Woodman CBJ, Luesley D,JordanJA. Previous cytology" in patients with invasive carcinoma of the cervix. Acta Gytol 1985;29:108-10. 10. Rylander E. Negative smears in women developing invasive cervical cancer. Acta Obstet Gynecot Scand 1977;56:115-8. 11. Berkowitz RS, Ehrmann RL, Lavizzo-Mourey R, Knapp PC. Invasive cervical carcinoma in young women. Gynecol Oncol 1979;8:311-6. 12. Paterson MEL, Peel KR, Joslin GAF.Cervical smear histories of 500 women with invasive cervical cancer in Yorkshire. BMJ 1984;289:896-8. 13. Wain GV, Farnsworth A, Hacker NF. Cervical carcinoma after negative pap smears: evidence against rapid-onset cancers. IntJ G?a~ecolCancer 1992;2:318. 14. Kristensen GB, Skyggebjerg K-D, Holund B, Holm K, Hansen MIC Analysis of cervical smears obtained within three years of the diagnosis of invasive cervical cancer. Acta Cytol 1991;35:47-50. 15. Peters RK, Thomas D, Skultin G, Henderson BE. Invasive squamous cell carcinoma of the cervix after recent negative cytologic test results--a distinct subgroup? Am J Obstet Gynecol 1988;158:926-35. 16. Walker EM, Hare MJ, Cooper R A retrospective review of cervical cytology in women developing invasive squamous cell carcinoma. BrJ Obstet Gynecol 1983;90:1087-91. 17. Morell ND, Taylor JR, Snyder RN, Ziel HK, Saltz A, Willie S. False-negative cytology rates in patients in whom invasive cervical cancer subsequently developed. Obstet Gynecol 1982;60:41-5.
Chicago, Illinois The Papanicolaou smear has been a remarkably effective tool in cancer prevention, but it is not a perfect test.Although the most important factor in failure of cervical cancer prevention is lack of adequate screening, other factors include problems with sampling, interpretation, and effective clinical follow-up. A small number of rapidly developing cervical cancers probably also arise in the interval between Papanicolaou smear screenings. Consequently, cervical cancer will develop in some women despite appropriate screening. This article will analyze some of the problems relating to diagnostic errors, which include abnormal cells that are few (<100), small, or bland. (Am J Obstet Gynecol 1996;175:1110-3.)
Key words; False-negative Papanicolaou smear, cervical carcinoma
Although no better cancer prevention test has ever been devised, Papanicolaou smear interpretation has been shown in many studies to be associated with a significant false-negative rate, ranging from 0% 1up to 94% 2 in 13 published trials (Table I). No cytology laboratory, however well run, is completely i m m u n e to such errors. Cervical cytologic analysis c a r r i e s with it an irreducible diagnostic error rate of at least 5% to 10% (i.e., at least 1 in every 10 to 20 positive cases will be missed in routine screening)? Other problems are also associated with use of the Papanicolaou smear, such as sampling errors and, more important, failure of women to be adequately screened. This report describes and evaluates some of the causes of interpretation errors resulting in false-negative diagnoses in cervical cytologic analysis.
whom high-grade cervical dysplasia or cervical carcinoma subsequently was diagnosed. The interval between the last negative report and the diagnosis ranged from an average of 9.3 months 2 to as long as 19 years4; in most cases, intervals were 5 years or less. The proportion of s m e a r s reclassified from "normal" to "abnormal" r a n g e s from 0% ~ to 94%2; with only two exceptions, however, at least a quarter of the cases analyzed were reclassified. Of the total 655 reported smears, 340, or about half (51.9%), were reclassified as abnormal. The definition of abnormal, however, ranged from atypical squamous cells of u n d e t e r m i n e d significance to malignant cells. This large proportion of reclassified smears indicates an extremely high false-negative rate. False-negative studies
Rescreening "negative" cervical smears preceding a "positive" diagnosis False-negative cervical cytologic reports are significant for at least three reasons: first, and most important, they represent the possibility that serious or even life-threatening disease has gone undetected and therefore untreated; second, they can provide an opportunity to evaluate whether a "rapidly progressive" form of cervical cancer truly exists or whether cases so diagnosed are, in fact, related to prior false negatives; and third, a study of false negatives can reveal areas where e r r o r s are most likely to be made and where corrective action might be possible. Table I shows results from 13 published rescreening studies involving women with prior negative smears in From the Section of Cytopathology, University of Chicago. Reprint requests: Richard M. DeMay, MD, Professorof Clinical Pathology, Direct~ Section of Cytopathology, University of Chicago, MARP 212, MC 2050, 5841 S. Maryland Ave., Chicago, IL 60637. Copyright © 1996 by Mosby-Yea,rBook, Inc. 0002-9378/96 $5.00+ 0 6/0/75667 1110
Hatem and Wilbur (1995) reviewed 17 "negative" smears immediately preceding a Papanicolaou smear showing high-grade squamous intraepithelial lesions or cervical carcinoma. 2 The time interval between the negative and positive smears averaged 9.3 months. The authors found that only 1 (6%) of 17 smears represented true negatives and, hence, possible rapid progression. The remaining 16 (94%) of 17 smears were considered to be false negatives. In 12 (75%) of these 16 cases, the s m e a r s contained fewer than 100 abnormal cells, making detection difficult. Most of these patients (88%) had histories of abnormal Papanicolaou smears. The missed lesions were analyzed: 6% were reclassified as "atypical cells of u n d e t e r m i n e d type and origin," 31% as "atypical cells" or "slight dysplasia," 31% as "highgrade squamous intraepithelial lesions," and 69% as "atypical immature squamous metaplastic cells" (some cases carried multiple diagnoses). The atypical immature squamous cells were described as very small cells, with high nuclear/cytoplasmic ratios, hyperchromatic nuclei, and occasionally irregular nuclear membranes and were
Volume 175, Number 4, Part 2 AmJ Obstet Gynecol
DeMay 1111
Table I. False-negative smears in cervical cytologic analysis "Negative" smears reclassified as "abnormal" Investigator (year)
''Negative" cytologic findings preceding "positive"
"'Negative" smears reviewed (n)
No.
%
Hatem and Wilbur (1995) 2 Attwood et al. (1985) 0 Robertson and Woodend (1993) 5 Rylander (1977) 10 Berkowitz et al. (1979) !~ Paterson et al. (1984) 12 Wain et al. (1992) 13 Sherman and Kelly (1992) 4 Kristensen et al. (1991) 14 Peters et al. (1988) 15 Walker et al. (1983) 16 Morell et al. (1982) 17 Liu (1967) 1
Averaged 9.3 mo Within 5 yr Up to 12 yr 4 to 5 )Jr Within 2 yr Within 10 yr Within 2 yr Median 93.5 mo Within 3 yr Within 3 yr Within 5 yr Within 3 yr 3.4yr
17 28 139 56 13 58 30 123 96 32 11 36 16 655
16 20 92 35 8 34 16 65 39 10 3 2 0 340
94.1 71.4 66.2 62.5 61.5 58.6 53.3 52.8 40.6 31.3 27.3 5.6 0 51.91
TOTAI~S
frequently f o u n d isolated in the smears. These cells resembled histiocytes or other b e n i g n cells. Harem a n d Wilbur 2 c o n c l u d e d that errors of screening or interpretation were i m p o r t a n t causes of false-negative reports in this p o p u l a t i o n a n d that these errors were related to the presence of few a b n o r m a l cells or small a b n o r m a l cells. O f clinical importance, however, is that patients with false-negative Papanicolaou smears usually have a history of a b n o r m a l smears. S h e r m a n a n d Kelly (1992) reviewed a total of 123 "negative" smears from 20 w o m e n in whom biopsyproved grade 3 cervical intraepithelial neoplasia (18 cases) or invasive cervical carcinoma (2 cases) developed, 4 The disease developed after three or m o r e "negative" Papanicolaou smears, with a m e d i a n interval of 93.5 m o n t h s (range 48 to 229 months). O n review, 23% of these smears were true negatives. A n o t h e r 25% of cases were reclassified as limited adequacy or completely unsatisfactory for evaluation (in 11% there was n o atypia, b u t the endocervical c o m p o n e n t was inadequate; 14% were unsatisfactory); 30% were reclassified as at)3oical squamous cells of u n d e t e r m i n e d significance; a n d 22% (28 smears) were considered false negatives, i n c l u d i n g 8% low-grade squamous intraepithelial lesions, 12% highgrade squamous intraepithelial lesions, a n d 2% squamous intraepithelial lesions of i n d e t e r m i n a t e degree. Analysis of these 28 false-negative smears showed that 14 (50%) c o n t a i n e d only a few (<100) cells diagnostic of squamous intraepithelial lesions. In five cases diagnostic cells were obscured by inflammation, a n d in four cases the a b n o r m a l cells were primarily present in tissue fragments or sheets that were difficult to distinguish from reactive or atrophic changes. Five false-negative smears involved small grade 3 cervical intraepithelial neoplasia cells with high nuclear/cytoplasmic ratios, irregular n u c l e a r outlines, a n d m i n i m a l hyperchromasia. These cells resembled cells of squamous metaplasia. Clinically, slightly more t h a n half the patients (55%)
had r e c u r r e n t Trichomonas infections. Approximately o n e third (35%) had unsatisfactory smears that had b e e n inappropriately reported as negative. O n e patient in four had a lapse in screening of 2 or more years. Interestingl); most patients (89%) had predominately endocervical lesions that were frequently located high in the endocervical canal. Even though an endocervical c o m p o n e n t was present in the smear, sampling of the lesion was still considered poor. Only 35% of patients had smears diagnostic of low-grade squamous intraepithelial lesions; all other a b n o r m a l smears were either atypical squamous cells of u n d e t e r m i n e d significance or high-grade squamous intraepithelial lesions. The authors c o n c l u d e d that most patients only appeared to have repeatedly negative Papanicolaou smears, when in reality most were either unsatisfactory for evaluation or extremely difficult to screen. 4 A long history of negative smears, especially in patients with multiple infections or heavy inflammation, should not, said the authors, be taken as proof of absence of disease. Many of the false negatives in this study were related to the presence of few diagnostic cells in the smear; most of these lesions were high in the endocervix a n d poorly sampled, despite the presence of an endocervical c o m p o n e n t . Problems in cytologic interpretation were also related to small, abnormal grade 3 cervical intraepithelial neoplasia cells that resembled squamous metaplasia. The description of these cells is similar to that of the cells in H a t e m a n d Wilbur's article s a n d characterizes cellular morphologic features that are particularly problematic for cervical cytologic screening. The differential diagnosis of tissue fragments can be also problematic in some cases; fragments derived from significant cervical lesions can be difficult to distinguish from b e n i g n processes such as atrophy or reactive changes. Finally the authors concluded that atypical squamous cells of u n d e t e r m i n e d significance may be the only identifiable cytolo~c abnormality d u r i n g the early d e v e l o p m e n t of high-grade squa-
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DeMay
mous intraepithelial lesions. Because of false negatives, reports of rapidly progressing cervical cancers should be viewed cautiously. A third study addressed the issue of false negatives in cervical cytologic testing. 5 The investigators reviewed 139 negative smears from 102 women, o b t a i n e d up to 12 years before a diagnosis of cervical cancer. The diagnoses included squamous cell carcinoma (83) a n d adenocarcin o m a (20). Review showed that only 47 (34%) of these 139 smears were true negatives a n d that most of these (64%) were deficient with respect to the endocervical c o m p o n e n t . The r e m a i n i n g 92 smears (66%) were false negatives, i n c l u d i n g 74 squamous cell carcinomas a n d 18 adenocarcinomas. In analysis of the false negatives, more than a quarter of cases (26 of 92 smears, or 28%) were characterized by the presence of few a b n o r m a l cells; most of these cases (18 of 26, or 69%) had a n inadequate endocervical c o m p o n e n t ; a n d of the r e m a i n i n g false negatives 7 of 26 had i n f l a m m a t o r y or postmenopausal changes. A n o t h e r quarter of the smears (24 of 92) had a b n o r m a l ceils present mainly as tissue fragments, with few single cells. This finding occurred in cases of both squamous a n d glandular abnormalities. Nearly half of the cases (42 of 92) had m o d e r a t e n u m b e r s or many abnormal cells present o n review. The authors c o n c l u d e d that smears with few a b n o r m a l cells are unlikely to be detected in routine screening. 5 Tissue fragments, or"microbiopsies," may be associated with either squamous or glandular neoplasia, a n d screeners are n o t sufficiently aware of the diagnostic i m p o r t a n c e of these fragments. I n f l a m m a t o r y a n d postmenopausal changes make diagnosis more difficult. In addition, a n inadequate endocervical c o m p o n e n t is associated with false negatives. Almost half of these false-negative cases contained n u m e r o u s a b n o r m a l cells o n review, yet screeners still failed to detect the a b n o r m a l cells. An i m p o r t a n t cause of false negatives was d e e m e d to be the h u m a n factor: screener fatigue. The same research group studied 62 smears from 62 patients with cervical carcinoma. 6 They reviewed findings in the earliest smear originally diagnosed as showing any abnormality. The smears were taken u p to 18 years before the diagnosis of cervical cancer: 60 patients had sqnamous cell carcinoma; two patients had adenocarcinoma. T h e majority of these smears were j u d g e d to be false negatives b u t also i n c l u d e d true positives in patients lost to follow-up. O n review, 51 (82%) of the 62 smears showed definite severe dyskaryosis (the British term for dysplasia) or grade 3 cervical intraepithelial neoplasia. A n o t h e r 6, or 10%, showed probable severe dyskaryosis; in these cases, smears c o n t a i n e d few a b n o r m a l cells or indicated inflammation. The r e m a i n d e r showed moderate dyskaryosis (4) or h u m a n papillomavirus-associated changes (1). I n n o case was mild dyskaryosis (mild dysplasia) diagnosed in a patient.
October 1996 AmJ Obstet Gynecol
The authors concluded that mild dyskaryosis (i.e., mild dysplasia) is only rarely a f o r e r u n n e r of invasive carcinoma. 6 Therefore, cancer prevention d e p e n d s primarily o n the detection a n d t r e a t m e n t of severe dyskaryosis (severe dysplasia) or grade 3 cervical intraepithelial neoplasia. These findings would support newer concepts that cervical carcinoma generally arises from an aggressive grade 3 cervical intraepithelial neoplasia lesion, widely present in the cervix a n d established years before invasion, rather than from the progression of low-grade cervical intraepithelial neoplasia. T h e authors' findings support the Bethesda System n o m e n c l a t u r e of low- a n d highgrade squamous intraepithelial lesions. 7 The final report to be discussed addresses screening results in the n o r m a l screening e n v i r o n m e n t compared with a test screening environment. 8Archival material consisted of five "false-negative" smears (smears initially diagnosed as n o r m a l b u t later f o u n d to contain cancer cells) a n d five "u-ue-positive" (smears diagnosed as mal i g n a n t from patients with cancer). The "false-negative" smears were considered diagnostically difficult cases; the "true-positive" smears, diagnostically straightforward. The five prior false-negative smears were tested with five screeners u n d e r n o r m a l screening cofiditions, without the screener's knowledge (by adding the cases in a b l i n d e d fashion to the daily workload of routine screening). The five prior true positives were similarly tested with five screeners u n d e r n o r m a l screening conditions, without the screener's knowledge. T h e n the false negatives were separately tested, b u t this time u n d e r test conditions, that is, with the screeners b e i n g forewarned that the routine cases were seeded with a b n o r m a l Papanicolaou smears. U n d e r n o r m a l screening conditions (i.e., the screeners were unaware they were being tested), only o n e correct detection out of a possible 25 was made (5 false-negative slides times 5 screeners). Even this single detection was a fluke, because the screener actually f o u n d an incompletely removed m a r k i n g dot rather than the m a l i g n a n t cells themselves. In four additional cases, the cases were called a b n o r m a l b u t n o t cancer. All true-positive cases were correctly identified o n the first review. In the second part of the test, in which the screeners were aware that they were being tested, the detection rate was considerably better; cancer was detected in seven of eight prior false-negative smears. However, this high detection rate occurred only at the expense of a threefold rise in the n u m b e r of smears identified as "suspicious" or "positive" a n d a d o u b l i n g of screening times. O n analysis, the false-negative smears were f o u n d to differ from true-positive smears in several ways. The falsenegative smears c o n t a i n e d few or no large tissue fragments; there were few or n o single a b n o r m a l cells; a n d the a b n o r m a l cells were small, with small nuclei, little pleomorphism, a n d m i n i m a l hyperchromasia. The authors c o n c l u d e d that it is virtually impossible to avoid all
Volume i75, Number 4, Part 2 AmJ Obstet Gynecol
false negatives. 8 The tedium of screening leads to false negatives, b u t u n d e r conditions of extreme alertness, although the false-negative rate is diminished, it comes at the expense of increased false positives a n d screening times and, therefore, diminished laboratory productivity. Comment
The five articles discussed provide examples of the typical cytodiagnostic problems in routine screening that lead to false-negative reports. Chief a m o n g these are smears c o n t a i n i n g few a b n o r m a l cells: detection of abnormality in slides with fewer than 100 a b n o r m a l cells was markedly poorer than in slides with m a n y such ceils. It is possible that 100 a b n o r m a l cells represent a diagnostic threshold below which detection is unreliable. The problem becomes c o m p o u n d e d when the a b n o r m a l cells are small a n d therefore n o t only difficult to detect b u t difficult to interpret once detected. These small a b n o r m a l cells can resemble ordinary i m m a t u r e metaplastic ceils or even histiocytes. 3 The diagnostic d i l e m m a posed by tissue fragments, or "hyperchromatic crowded groups" has n o t b e e n sufficiently emphasized. 3 Hyperchromatic crowded groups are the general morphologic appearance of a variety of serious lesions in the Papanicolaou smear, including in situ or invasive carcinoma, b u t similar groups are also associated with b e n i g n entities, such as atrophy or e n d o m e t r i a l cells. The differential diagnosis of hyperchromatic crowded groups can be very difficult in some cases. Abnormality of glandular cells can be even more difficult to accurately identify a n d diagnose than squamous abnormalities. High-grade, small-cell lesions located high in the endocervical canal are particularly likely to evade detection. In addition, atypical squamous cells of u n d e t e r m i n e d significance are an i m p o r t a n t diagnosis a n d may be the only abnormality detectable in the Papanicolaou smear preceding the d e v e l o p m e n t of high-grade squamous intraepithelial lesions. Most patients with false-negative Papanicolaou smears have histories of a b n o r m a l smears, b u t multiple "negatives" do n o t completely exclude a clinically significant lesion. A n o t h e r i m p o r t a n t cause of app a r e n t false negatives is limited or unsatisfactory smears that are n o t properly identified as such. Finally, although rapid progression of cervical carcinoma may occur, m a n y cases represent prior false-negative diagnoses. In summary, false negatives are inevitable in routine cervical cytologic screening. Patients a n d clinicians alike should be w a r n e d that n o cytology laboratory is completely free of diagnostic errors; errors occur at a low b u t well-docmnented a n d probably irreducible rate of at least 5% to 10% in r o u t i n e screening. 3 Unfortunately; this means that cervical carcinoma will develop in some w o m e n despite appropriate screening. To reduce the risk of this tragic outcome, w o m e n should obtain regular Papanicolaou smears even if they have long histories of n o r m a l
DeMay 1113
cervical cytologic findings. All a b n o r m a l results, including a diagnosis of atypical squamous ceils of u n d e t e r m i n e d significance, should be followed up as clinically appropriate with, of course, biopsy of any visible suspicious lesions a n d investigation of any suspicious symptoms. Finally, it is i m p o r t a n t when j u d g i n g the Papanicolaou smear to realize that, although it is imperfect, n o other test has b e e n as successful in preventing a n d detecting cancer. W o m e n are more likely to have cervical cancer related to inadequate screening than to misdiagnosed Papanicolaou smears?
REFERENCES
1. Liu W. Positive smears in previously screened patients (certain cytologic findings of public health importance). Acta Cytol 1967;11:193-8. 2. Harem F, Wilbur D. High-grade cervical lesions following negative Papanicolaou smears: false-negative cervical cytology or rapid progression. Diagn Cytopathol 1995;12:13541. 3. DeMay RM. Failure of the Pap smear. In: The art & science of cytopathology. Chicago: ASCP Press, 1996:141-7. 4. Sherman ME, Kelly D. High-grade squamous intraepithelial lesions and invasive carcinoma following the report of three negative Papanicotaou smears: screening failures or rapid progression? Mod Pathol 1992;5:337-42. 5. RobertsonJH, Woodend B. Negative cytology preceding cervical cancer: causes and prevention..] Clin Pathot 1993;46: 700-2. 6. Robertson JH, Woodend B, Elliott H. Cytological changes preceding cervical cancer. J Clin Pathol 1994;47:278-9. 7. Sherman ME, Schiffman MH, Erozan YS, Wacholder S, Kurman RJ. The Bethesda System. A proposal for reporting abnormal cervical smears based on the reproducibility of cytopathologic diagnoses, Arch Pathol Lab Med 1992;116: 1155-8. 8. Bosch MMC, Petronella ElVl, Rietveld-Scheffers CT, Boon ME. Characteristics of false-negative smears tested in the normal screening situation. Acta Gytol 1992;36:711-6. 9. Attwood ME, Woodman CBJ, Luesley D,JordanJA. Previous cytology" in patients with invasive carcinoma of the cervix. Acta Gytol 1985;29:108-10. 10. Rylander E. Negative smears in women developing invasive cervical cancer. Acta Obstet Gynecot Scand 1977;56:115-8. 11. Berkowitz RS, Ehrmann RL, Lavizzo-Mourey R, Knapp PC. Invasive cervical carcinoma in young women. Gynecol Oncol 1979;8:311-6. 12. Paterson MEL, Peel KR, Joslin GAF.Cervical smear histories of 500 women with invasive cervical cancer in Yorkshire. BMJ 1984;289:896-8. 13. Wain GV, Farnsworth A, Hacker NF. Cervical carcinoma after negative pap smears: evidence against rapid-onset cancers. IntJ G?a~ecolCancer 1992;2:318. 14. Kristensen GB, Skyggebjerg K-D, Holund B, Holm K, Hansen MIC Analysis of cervical smears obtained within three years of the diagnosis of invasive cervical cancer. Acta Cytol 1991;35:47-50. 15. Peters RK, Thomas D, Skultin G, Henderson BE. Invasive squamous cell carcinoma of the cervix after recent negative cytologic test results--a distinct subgroup? Am J Obstet Gynecol 1988;158:926-35. 16. Walker EM, Hare MJ, Cooper R A retrospective review of cervical cytology in women developing invasive squamous cell carcinoma. BrJ Obstet Gynecol 1983;90:1087-91. 17. Morell ND, Taylor JR, Snyder RN, Ziel HK, Saltz A, Willie S. False-negative cytology rates in patients in whom invasive cervical cancer subsequently developed. Obstet Gynecol 1982;60:41-5.