Investigation of 100 consecutive negative cone biopsies

British Journal of Obstetrics and Gynaecology January 1997, Vol. 104, pp. 100-104 Investigation of 100 consecutive negative cone biopsies *Pouran Golbang Research Fellow and Consultant (Gynaecology), **James Scurry Consultant (Histopathology), **Sarah de Jong Medical Scientist, * *Dominique McKenzie Cytologist, *Robert Planner Consultant (Gynaecology),?Jan Pyman Consultant (Histopathology), TtRuth Davoren Consultant (Histopathologist) Departments of +Oncology and **Pathology, Mercy Hospital for Women, t Kctorian Cytology Service and YfCytopath Histology and Cytologv, Melbourne, Australia Objective To investigate the reasons for cone biopsies reported as not containing intraepithelial or invasive malignancy and thereby find ways to decrease their incidence. Design One hundred cone biopsies reported as negative were identified out of a total of 436 consecutive cone biopsies. The patients cytology, colposcopy and histology reports and cytology and histology slides were reviewed. Further opinions in cases of doubt were obtained in cytology and histology. In cone biopsies still considered negative after reviews, deeper levels were cut, exhausting all paraffin blocks. Follow up cytology, colposcopy and histology were reviewed. Setting Gynaecological oncology unit in a university teaching hospital. Results After re-evaluation the final diagnoses of cone biopsies initially reported as negative were positive (n = 21), unsatisfactory (n = 27) and true negative (n = SI), with one case excluded because of insufficient material for review. The positive cases were diagnosed on review (n = 11) or extra levels (n = 10). The unsatisfactory cases were all due to denudation. The 5 1 true negative cases were divided into those which never had had histologic confirmation by punch biopsy or endocervical curettage (n = 47) and those with a previously confirmed histological abnormality (n = 4). Conclusions The number of negative cone biopsies can be reduced by: 1. taking Pap smears after correction of atrophy and inflammation; 2. more scrupulous colposcopy aimed at reducing the number of unsatisfactory colposcopies or misinterpreted colposcopic findings; this thorough examination should include the vagina and vulva; 3. confirmation of smear and colposcopic findings by biopsy prior to cold-knife conisation and performing a large loop excision of the transformation zone (LLETZ) for cases where there is a discrepancy between the smear abnormality and colposcopy/biopsy findings; 4. good quality cone biopsies using a technique that does not handle the mucosa and is performed after the mucosa has had time to regenerate following the colposcopic investigations; and 5. exhausting all blocks with multiple levels before reporting a cone biopsy as negative. INTRODUCTION The traditional management of preinvasive disease of the cervix begins with an abnormal smear detected on routine screening. The patient is referred to a colposcopist who attempts to grade, delineate and biopsy the abnormality. A colposcopic-directed biopsy confirms the smear and colposcopic diagnoses and, at a second visit, the abnormal transformation zone is destroyed. Where there is suspicion of invasion or adenocarcinoma in situ or an unsatisfactory colposcopy (defined as an inability to see the full transformation zone, the endocervical limits of a lesion or a discrepancy between the smear and colposcopic findings) a cone biopsy is performed. Correspondence: Dr J. Scurry, Department of Pathology, Mercy Hospital for Women, Clarendon Street, East Melbourne, Victoria 3002, Australia. The cone biopsy usually provides a definitive diagnosis, with type, grade, extent of the lesion and state of the resection lines. Not infrequently, however, no lesion is found on histological examination of the cone biopsy. There are many potential reasons for negative cone biopsies, including misreporting of cytology, colposcopy and/or histology, denudation of the mucosa or abnormal cells translocated from elsewhere in the genital tract accounting for the original smear diagnosis. Negative cone biopsies are unsatisfactory to both clinician and patient. While one important benefit is the virtual exclusion of invasive cervical cancer, the abnormal cytology still has to be explained, and difficulties can develop during follow up because of stenosis of the endocervical canal, particularly in postmenopausal women. Further operations, such as dilatation of the endocervical canal and/or hysterectomy, should be considered. While cone biopsy 100 0 RCOG 1997 British Journal of Obstetrics and Gynaecology

NEGATIVE CONE BIOPSIES 101 is a relatively safe procedure, there are possible complications of excessive haemorrhage, general anaesthetic risks and cervical incompetence with future pregnancy losses. In recent years the loop biopsy has replaced cold-knife conisation for certain indications and has the advantage that it can be performed under local anaesthetic, however, a negative loop biopsy poses similar problems in management to a negative cold knife cone biopsy. Although there have been studies reporting the incidence of negative cone biopsies as 45% to 64% depending on the indication for cone biopsy, there is only one published study of which we are aware that investigates negative cone biopsies4. Luesley et al. compared the results of pre-cone investigations in patients with positive and negative cone biopsies and found that a colposcopic suspicion of invasion, a positive pre-cone smear, two severely dyskaryotic smears within a 12 month period, previous abnormal histology and previous treatment for cervical intraepithelial neoplasia could be used to predict the presence of intraepithelial neoplasia or invasive malignancy in the cone biopsy. Conversely, 44% of negative cones could be avoided without overlooking any cases of micro- or occult invasive carcinoma. We have reviewed all cytology, histology and follow up and, where relevant, cut deeper levels of histology blocks in 100 consecutive patients with reported negative cone biopsies in an effort to determine the reasons for the negative result and thereby make recommendations on how their incidence can be reduced in the future. METHODS The reports of all patients who underwent a coldknife cone biopsy at the Mercy Hospital for Women beginning January 1989 were examined to locate 100 consecutive negative cone biopsy reports. Reports were classified as positive if they contained intraepithelial or invasive neoplasia and negative if they did not. By August 1994, after examining 436 reports, 100 negative cone biopsy reports were identified, giving a crude negative cone rate of 23%. All available cytology reports and slides, colposcopy reports, histology reports and slides of each patient with a negative cone biopsy report were reviewed. During the period of study, the standard protocol had been to open the cone biopsy in the midline anteriorly, slice it into multiple blocks 2-3 mm thick and cut three levels at 200 pm intervals from each block. The review process was performed by pathologists and cytologists who knew that they were reviewing cases in a negative cone study. There were no positive or negative controls in the review Table 1. Protocol for follow up of cone biopsies. For patients with negative cone biopsies the three-month visit includes an extra colposcopy to exclude vaginal abnormality. After three negative Pap smears, patients revert to an annual Pap smear. Time Procedure 6 weeks Routine post-operative visit 3 months Pap smear 6 months 12 months Colp and Pap smear, biopsy if indicated Pap smear process. Intra-observer variation in cytology or histology was not able to be assessed as the smears and cone biopsies were largely reviewed by different personnel than originally reported. The following information was recorded for each patient: 1. The original and review cytological prediction. 2. The colposcopic findings. 3. The original and review diagnoses of all target biopsies, endocervical and uterine curettings and cone biopsies. 4. Further opinions on the cone biopsies with difficulties in diagnosis. The paraffin blocks of the cone biopsies that were still considered negative after review and second opinion were then cut into multiple further levels, also at 200 pm intervals, until all blocks were exhausted and these extra levels examined. In cases that were still negative after these steps, it was noted in the cone biopsy whether the transformation zone was present and whether there was significant denudation or other factors hampering interpretation. The follow up cytology, colposcopy and histology reports and slides were reviewed for each case. Patients who have a cone biopsy at Mercy Hospital are followed up according to the protocol shown in Table 1. At the conclusion of the review process the cone biopsies were given a final diagnosis ofpositive if intraepithelial or invasive neoplasia was found on review, further opinion or deeper levels, unsatisfuctory if the entire squamocolumnar junction was not present in the slides or negative if none of the above were found. RESULTS The final diagnoses of the cone biopsies after investigations are given in Table 2. Note that one case was excluded because of lack of material for review. Positive There were 2 1 positive diagnoses. Inter-observer variation of opinion on the histology of cone biopsies occurred in 11 cases: in five patients the reviewing

102 P. GOLBANG ET AL Table 2. Final histological diagnoses of 100 negative cone biopsies after review and deeper levels. One case was excluded because of a lack of material for review, therefore total n = 99. Positive Unsatisfactory Diagnosis changed Denudation on review (n = 5) (n = 27) Diagnosis changed on second opinion (n = 6) Diagnosis changed on extra levels (n = 10) n=21 n=27 True Negative Non-histologically confirmed pap smear abnormality (n = 47) Histologically confirmed abnormality (n = 4) Abnormality on follow up (n = 3) n=51 pathologist (J.S.) and in six the second opinion pathologist (R.D.) reclassified the cone biopsies as showing intraepithelial neoplasia. Difficulties distinguishing atrophic, inflammatory and/or metaplastic changes from intraepithelial neoplasia accounted for the differences in opinion between pathologists in all cases. Between three and eight extra levels (mean extra 4.2 levels) were cut on each block of each cone biopsy that was still negative after review and second opinion. Examination of these extra levels led to the diagnosis of another ten cases with intraepithelial neoplasia. Unsatisfactory There were 27 women whose cone biopsies showed denudation involving the squamocolumnar junction in at least one block, and nearly all of these had denudation involving multiple blocks. This epithelial stripping was particularly related to conisation using a technique which involved probing the endocervical canal. Opening the unfixed cone biopsy in the laboratory may have been another factor. True negative The 51 cone biopsies which remained negative after reviews and extra levels and had a full covering of epithelium for assessment were called true negative cones. In this group, the age range was 24 to 72 years (mean 50.6); menopausal status was not recorded. The duration of the cytological abnormality before conisation was less than six weeks in 8 patients, six weeks to three months in 11, greater than three months in 13 and not known in 19. The interval between colposcopy and cone biopsy was less than six weeks in 32 patients, six weeks to three months in 5, greater than three months in 3 and not known in 11. The true negative cone patients were divided into thqse which had a histologically confirmed abnormality (n = 4) and those that did not (n = 47). True negative cone biopsies with histologically confirmed abnormality Three of the four patients with negative cone biopsies had small lesions seen at colposcopy and confirmed high grade cervical intraepithelial neoplasia (CIN) on biopsy, suggesting that the biopsy removed the CIN before the cone biopsy. Another patient with referral and colposcopic smear showing CIN 2, had a normal colposcopy, but CIN 1-2 on endocervical curetting, again suggesting that the abnormality was removed by curettage. True negative cones without histologically confirmed abnormality The cone biopsies in these 47 patients were performed on the basis of an abnormal smear and a positive or unsatisfactory colposcopy. The original diagnoses of the first smear abnormality were high grade abnormality in 23 women, low grade or negative in 15 and inconclusive in two. The results of smears in seven women were not available. These referral smears were available in 23 women for review. The review diagnoses were high grade abnormality in six women, low grade or negative in ten and inconclusive in seven. Colposcopic smears were reviewed in 31 patients. The review diagnoses were high grade abnormality in only six women and low grade or negative in 25. Of 39 colposcopies whose results were available for review, high grade abnormalities were diagnosed in only eight. Biopsies and endocervical curettage were performed in 21 women, and none had proven high grade abnormality. The cone biopsies in 18 patients showed benign changes (e.g. atrophy, inflammation, immature metaplasia, basal cell hyperplasia and HPV) that were difficult to distinguish from intraepithelial neoplasia. When the original Papanicolaou (Pap) smears were reviewed in conjunction with these 18 cone biopsies, the benign changes at least partly explained the Pap smear findings. One woman had a history of carcinoma of the breast; she had a cone biopsy for an abnormal looking cervix on colposcopy, despite a normal smear, but no abnormality was predicted in the cone biopsy and none was found. Follow up Eighty-eight patients were followed up at Mercy Hospital from six months to five years. At the time of writing, none of them has developed an invasive carcinoma of the cervix. A total of 276 smears, 87 colposcopies and 25 biopsy procedures have been performed in follow up. Of the patients with true 0 RCOG 1997 Br J Obstet GynaecoZlO4, 100-104

NEGATIVE CONE BIOPSIES 103 negative cone biopsies, two had vaginal intraepithelial neoplasia, one had CIN on repeat cone biopsy following further abnormal smears, and one had endometrial carcinoma who had had a glandular cell abnormality reported on original Pap smear. A fifth patient who had a positive cone biopsy also developed vaginal intraepithelial neoplasia and a sixth patient with a denuded cone biopsy had CIN diagnosed on follow up smear and confirmed histologically. DISCUSSION The crude negative cone rate in this study was 23%; however, after investigation and exclusion of positive and unsatisfactory cases, the true negative cone rate fell to 11.7%, which is comparable to the negative cone rates in other studies of random cone bi~psies~-~. Positive cases Inter-observer variation of opinion in 1 1 cases (1 1%) reflected difficulties for histopathologists in distinguishing atrophy, inflammatory change, metaplasia and basal cell hyperplasia from intraepithelial neoplasia. The finding of intraepithelial neoplasia in deeper levels in 10 cases (1 0%) indicates that a negative cone biopsy report should not be issued until deeper levels have been cut, exhausting all blocks. The practice of cutting three levels at 200 pm intervals examines less than one third of a 2 mm thick block. Unsatisfactory cases Denudation was found in 27 negative cone biopies preventing complete assessment of the transformation zone. It is incorrect to assume that a denuded specimen is normal as dysplastic epithelium is easily stripped at the time of conisation. Twenty-seven percent unsatisfactory specimens in a procedure that is essentially diagnostic is disappointingly high, but there are no figures in the literature with which we can compare this. The strict criterion for inclusion into this category of only one slide showing loss of epithelium at the squamocolumnar junction may account for the high number of unsatisfactory cases. Damage to the epithelium can occur before, during and after conisation. At colposcopy, rough handling, smear taking, endocervical curettage and biopsies all damage epithelium. Enough time must be given for the epithelium to heal between colpscopy and conisation. During conisation, probing the cervical canal before cutting the cone biopsy was a potent cause of denudation in our study. Rough handling of the specimen in the laboratory was another possible cause of denudation. The blocking of the cone specimen by the pathologist should involve a technique that does not handle the mucosa. We now fix the cone biopsy whole and cut the cone after fixation into sequential parasagittal slices 2-3 mm thick after painting the stromal resection lines and find this preferable to our previous technique of opening the cone in the fresh state, pinning it out on cork board, fixing it and then slicing it into multiple longitudinal blocks. True negative In this study a small area of abnormal epithelium would appear to have been completely removed by biopsy and/or endocervical curettage in four cases. There are a number of possible reasons why a biopsy-proven abnormality is not found in the cone biopsy including complete removal by biopsy, pregnancy, delivery and regressions. A much greater problem were the 47 patients with true negative cone biopsies who had not had their smear predictions confirmed as histological abnormalities. This group had a mean age of 50.6 years, significantly older than the mean age of 39 years reported for the group in the large study by Luesley et al4 of 899 patients with cone biopsies. Older women are liable to have atrophic changes on smears which could be confused with intraepithelial neoplasia as well as unsatisfactory colposcopy due to inversion of the squamocolumnar junction and atrophic changes in the mucosa. Furthermore, 68% had their cone biopsies less than six weeks after colposcopy. Performing a cone biopsy soon after colposcopy, if not denuding the transformation zone, gives the pathologist the problem of distinguishing degenerative and regenerative changes from intraepithelial neoplasia in the cone biopsy. In the patients with true negative cone biopsies, the minimum indication for cone biopsy was a smear abnormality with an unsatisfactory colposcopy. Misinterpretation of cytology could be a very important reason for the performance of negative cone biopsies. While there has been great emphasis on false negative smears, false positive smears have not attracted as much attention in the literature. A negative cone biopsy study is biased to show a high rate of false positive Pap smears. In this study the referral Pap smears of five patients with true negative cone biopsies were considered on review to be overcalled. Three were judged false positives and two downgraded from high grade to low grade abnormality. The reasons for overcall were atrophy, inflammation, metaplasia, high endocervical canal sample and

104 P. GOLBANG ET AL difficulty in diagnosing glandular lesions. The annual report of the Victorian Cervical Cytology Register (1994) compared the results of cytology and corresponding histology/ colposcopy findings. On cytology, 37% of biopsies were reported as normal for atypiahuman papillomavirus reported, for CIN 1 as 19%, for CIN 2 as 11% and for CIN 2-3 as 7%6. Although these statistics also suggest a high rate of false positive cytology, it should be kept in mind that the biopsies were target biopsies of an abnormal area found at colposcopy. Wrong site of sampling and poor sampling techniques could have contributed to these high rates. Adenocarcinoma in situ is a particular problem. In this study eight patients with true negative cones had a smear and/or colposcopic suggestion of adenocarcinoma in situ, the diagnosis of which can be difficult on Pap smear and colposcopy. If this is suggested on smear, patients generally proceed to cone biopsy irrespective of colposcopy and biopsy findings and the suspicion of this condition would appear to account for an unavoidable cause of negative cone biopsies. Colposcopy has greatly reduced the need for cone biopsy7, but unsatisfactory colposcopies are a major problem. Of the 42 colposcopies in the true negative group where the findings were available for review, 35 (82%) were unsatisfactory, comprising eight women whose transformation zone was not seen, 12 in whom the limits of an abnormality were not seen and 15 in whom there was a discrepancy between cytology and colposcopy or biopsy findings. Unsuccessful colposcopy is particularly prone to occur in older women in whom the transformation zone tends to retract into the endocervical canal. Successful colposcopy depends on the experience of the operator. Complete and careful examination with the use of special devices such as the endocervical and vaginal wall specula and the liberal use of acetic acid and iodine (in patients without atrophy) to mark abnormal areas will help achieve successful colposcopic examination. Even when the transformation zone can be fully visualised through the colposcope, atrophy due to menopause, postpartum status, and to a lesser extent long term use of oral contraceptives (especially progesterone-dominant types) and inflammation may be confused with a major abnormality. Stromal decidualisation in pregnancy may also cause diagnostic problems. Treatment of atrophy with oestrogen locally and/or systemically is important to reduce atrophy. Treatment of infectious conditions with appropriate antibiotics is also beneficial, although inflammation of the cervix is often not caused by treatable pathogens which respond to treatment. Awareness of changes associated with pregnancy also helps reduce diagnostic error in colposcopy, At colposcopy it is essential to examine all of the possible sites of origin of cytologic abnormality including the vagina and vulva. In two women with true negative cone biopsies in this study, vaginal intraepithelial neoplasia was found in follow up after a negative cone biopsy. Perhaps cone biopsy could have been avoided if vaginal intraepithelial neoplasia had been diagnosed at colposcopy. The most important messages to be read from this study are that the incidence of negative cone biopsies can be reduced by attention to detail at all steps in the work-up of a smear abnormality and that the patient most liable to have a negative cone is the older woman with an atrophic cervix, where smear interpretation is difficult and colposcopy unsatisfactory. References Moore EJ, Fitzpatrick CC, Coughlan BM. et al. Cone biopsy: a review of 112 cases. Ir Med J 1992; 85: 28-30. Lopes A, Pearson SE, Mor-Yosef S et al. Is it time for the reconsideration of the criteria for cone biopsy. Br J Obstet Gynaecol 1989; 96 1345-1 347. Howel R, Hammond R, Pryse-Davies J. The histologic reliability of laser cone biopsy. Obstet Gynecoll991; 77: 905-91 1. Luesley DM, Wade-Evans T, Jordan JA, Woodman CBJ. Negative cone biopsies and their prediction. Br J Obstet Gynaecol 1987; 94: 895-900. Fletcher A, Metaxas N, Grubb C et al. Four and a half year follow up of women with dyskaryotic cervical smears. BMJ 1990; 301: 641-644. Mitchell H, Higgins V. Victorian Cervical Cytologv Register. Statistical Report, 1994. Melbourne, Australia: VCCR, 1994. Benedict JL, Anderson G, H,, Simpson ML. et al. Colposcopy, conization and hysterectomy practices: A current perspective. Obstet Gynecoll982; 60: 539-545. Received 5 March I996 Accepted I5 July 1996