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Examination of Sources of Diagnostic Error Leading to Cervical Cone Biopsies With No Evidence of Dysplasia

Alison Carrigg DO, Crystal Teschendorf CT(ASCP), Deirdre Amaro MD, Noel Weidner MD, Ann Tipps MD, Ahmed Shabaik MD, Michael R. Peterson MDPhD, Grace Y. Lin MDPhD, Farnaz Hasteh MD
DOI: http://dx.doi.org/10.1309/AJCP6BSD0SNGQLHQ 422-427 First published online: 1 April 2013

Abstract

At our institution, 17% of cervical conization specimens are reported as negative for dysplasia or malignancy. To identify sources of error, we reviewed 53 negative conization specimens and their prior and follow-up cytology, biopsy, and endocervical curettage specimens. Examination of deeper-level sections and p16 immunostaining were performed on all conization specimens and selected biopsy specimens. Dysplasia was detected in 26% (14/53) of conization specimens. Twenty-eight percent (15/53) of cones were truly negative, and the presurgical material had been overcalled as high-grade squamous intraepithelial lesions (HSIL). Forty-five percent (24/53) of cones were truly negative and HSIL was confirmed in the presurgical material. Of these, 11% (6/53) showed subsequent evidence of residual dysplasia and 26% (14/53) were negative on further follow-up. Deeper-level sections, p16 immunostains, and consensus review may help identify squamous dysplasia in conization specimens and may prevent the overdiagnosis of HSIL on cervical biopsies.

Key Words
  • Cone biopsy
  • p16
  • Cervix
  • Diagnostic error

Cervical conization (including loop electrosurgical excision procedure, cold knife conization, and laser conization) is a common therapeutic modality used for cervical dysplasia. However, these procedures carry risks, including premature rupture of membranes, infertility, cervical stenosis, and hemorrhage.13

Cervical conization specimens with no evidence of dysplasia or malignancy are a relatively common occurrence, with negative cone rates ranging from 10.6% to 34% reported in the literature.47 Previous studies have identified useful immunostains for identifying occult dysplasia in cervical tissue.8,9 Chief among these is the tumor suppressor protein p16INK4A encoded by the CDKN2A gene. Immunohistochemistry for p16 is widely acknowledged as a sensitive and specific surrogate marker of dysplasia.10,11

To identify sources of potential error leading to negative conization specimens, we reviewed 53 cervical conization specimens initially diagnosed as negative for dysplasia from our archives, as well as preceding diagnostic specimens and follow-up data when available. We endeavored to determine the cause and outcomes of these initially negative cone specimens by using a combination of diagnostic tools: deeper-level sections, immunohistochemical stains for p16, and a consensus panel review.

Materials and Methods

A computer-generated search of our files identified 610 consecutive cervical cone biopsy reports from University of California San Diego Health System during a 3-year period from 2004 to 2007. One hundred six (17%) specimens showed no evidence of dysplasia or malignancy. Excluding all patients who did not undergo prediagnostic testing at our facility, 53 cone biopsy specimens were negative for dysplasia or malignancy despite a diagnosis of squamous intraepithelial lesion, grade cannot be determined (SIL) or high-grade squamous intraepithelial lesion (HSIL) on presurgical tests.

The slides for all the cervical cones as well as the presurgical Papanicolaou (Pap) smears and biopsy and endocervical curettage (ECC) specimens were reviewed by a panel of 3 pathologists (F.H., G.Y.L., and M.R.P.). Both positive and negative control cases for the cones, Pap smears, biopsy specimens, and ECCs were randomly selected from the same 3-year time frame. A total of 73 conization specimens, 66 Pap smears, 59 biopsy specimens, and 63 ECC specimens were reviewed. All slides were deidentified so that the reviewers were blind to patient information and initial diagnosis. In addition, the cases were unbatched so that the reviewers did not know which specimens came from a single patient.

The conization specimens were examined after a minimum of 3 levels was obtained from each tissue block. In the cases that did not reveal dysplasia on deeper levels, a p16 immunohistochemical stain (clone JC8, SC-5630, Santa Cruz Biotechnology [Santa Cruz, CA] at 1:80 dilution, rabbit polyclonal IgG antibody) was completed on the most atypical slide selected by the authors (F.H., G.Y.L., and M.R.P.).

A senior cytotechnologist (C.T.) and the pathologist panel also reviewed the presurgical Pap smears. Thirty-nine (59%) of the 66 Pap smears that were reviewed were conventional smears and the remaining 27 were SurePath Pap slides (BD, Franklin Lakes, NJ). After initial review of the presurgical cervical biopsy and ECC specimens, section levels (6 biopsies, 2 ECCs) and immunohistochemical staining for p16 (24 biopsies, 9 ECCs) were completed on all biopsy and ECC specimens that were deemed to require these special tests by at least 2 reviewers. For both surgical and presurgical cases in which a single diagnosis was not unanimously agreed upon, an additional review by 3 senior pathologists (A.S., A.T., and N.W.) was initiated and a consensus diagnosis was reached. Pathology follow-up data were available for 39 patients and ranged from 5 to 7 years.

Results

During a 3-year period, 106 (17%) of 610 conization specimens showed no evidence of dysplasia or malignancy. Excluding all patients who did not undergo prediagnostic testing at our facility, 53 cone biopsy specimens were negative for dysplasia or malignancy despite a diagnosis of SIL or HSIL on presurgical tests. The average age of all the patients in the study was 36 years (range, 18–86 years) and the average age for the control cases was 41 years (range, 19–78 years). The average age of the patients with confirmed HSIL or SIL on presurgical tests but a negative cone and negative results on follow-up was 30 years (range, 22–60 years).

The overall data are summarized in Figure 1. Of the 53 cervical conization specimens that were called negative initially, a total of 14 (26.4%) cases were found to have dysplasia that was missed at the initial evaluation. Three cases (5.7%) were identified after consensus review. Four cases (7.5%) were identified after review of an additional level section. The other half (13.2%) were found to have dysplasia on review of additional level sections as well as p16 immunohistochemical staining Image 1. Of these 14 cases with dysplasia, 6 cases showed low-grade squamous intraepithelial lesions (LSIL), 5 HSIL, and 3 SIL. Follow-up data were available for 6 of these patients: 1 woman was found to have HSIL on a subsequent Pap smear and 5 women had negative or atypical squamous cells of undetermined significance (ASCUS) on follow-up specimens Table 1.

Figure 1

Summary of findings of this study. ASC-H, atypical squamous cells, cannot exclude HSIL; ASCUS, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.

Image 1

Examples of conization specimens that were initially diagnosed as negative for dysplasia but upgraded to high-grade squamous intraepithelial lesion after additional level review and p16 performed at the time of the study. A, H&E, × 200; B, P16 immunohistochemical stain on the same conization specimen as panel A, × 200; C, H&E, × 100; D, P16 immunohistochemical stain on the same conization specimen as panel C, × 100.

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The remaining 39 (73.6%) conization specimens were confirmed to be negative after review of levels, p16 staining, and consensus review. The presurgical Pap smears, biopsy specimens, and/or ECC specimens were reviewed with positive and negative controls and were blinded and unbatched, so it could not be determined which cases were associated with one another and with which conization specimen. Review of the presurgical specimens confirmed the prior diagnosis in 24 cases but also revealed that presurgical specimens of 15 patients (28.3%) were overcalled initially. In 11 patients, a single specimen was misdiagnosed as HSIL, and in 4 patients, 2 presurgical specimens were misdiagnosed as HSIL Table 2. Eight Pap smears were downgraded: 1 case was downgraded from HSIL to benign, 2 cases from HSIL to ASCUS, 1 case from HSIL to ASC, cannot rule out high-grade lesion (ASC-H), 3 cases from HSIL to LSIL, and 1 case from LSIL to ASCUS Table 3. Ten cervical biopsy specimens were downgraded: 1 case was downgraded from HSIL to benign, and 9 cases were downgraded from HSIL to LSIL Table 4. In 7 of these biopsy specimens for which diagnosis of HSIL was downgraded, p16 immunostaining performed at the time of the study had negative results (see Image 2 for example). One ECC specimen was downgraded from HSIL to benign. Overall, in most cases, the most severe grade of dysplasia was downgraded from HSIL to LSIL in at least 1 of the presurgical specimens; however, in 2 patients, the most severe diagnosis on presurgical material was downgraded to ASCUS and in 1 patient to ASC-H (Figure 1). Follow-up data were available for 13 of the 15 women overdiagnosed on presurgical material: 1 patient whose presurgical material was downgraded from HSIL to LSIL had vaginal intraepithelial neoplasia 2 on follow-up, but the remainder of the patients had no dysplasia or ASCUS on follow-up (Table 1).

Image 2

Cervical biopsy specimen that was initially diagnosed as high-grade squamous intraepithelial lesion without the use of p16 immunohistochemistry. Subsequent immunostaining for this study revealed this case to be p16 negative, resulting in a downgrade of the diagnosis to benign. A, H&E, × 100; B, P16 immunohistochemical stain, × 100.

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Follow-up data were available for 20 of the 24 patients with confirmed HSIL on presurgical specimens and confirmed negative conization specimens (Figure 1, Table 1). In 6 patients (11.3%), dysplasia or carcinoma was identified on follow-up specimens. One woman had multiple subsequent specimens with HSIL, including ECC and endometrial biopsy specimens, and eventually developed invasive squamous cell carcinoma. We did not consider a single case in which the patient was diagnosed with LSIL 6 years after the cone biopsy to be a positive follow-up result because of the possibility of reinfection. The remaining women had either no dysplasia or ASCUS on follow-up. The women with positive follow-up results had an average age of 43 years (range, 25–59 years). The remaining women with negative follow-up results had an average age of 31 years (range, 22–51 years).

Because most of the Pap smears were initially diagnosed as ASC-H, LSIL, or HSIL, human papillomavirus (HPV) testing was not performed at the time. Only 9 of the 53 patients underwent concurrent high-risk HPV testing at the time of Pap smear, biopsy, or conization. Eight of the 9 patients were high-risk HPV positive. However, because of the small numbers, we could not further analyze the significance of the high-risk HPV status of these patients.

Discussion

Cervical conization specimens with no evidence of dysplasia or malignancy are a relatively common occurrence, with negative cone rates reported in the literature ranging from 10.6% to 34%.47,12 A computer-generated search of our files identified that 106 (17%) of 610 consecutive cervical cone biopsies were reported as negative. Excluding patients who did not receive presurgical diagnostic testing at our facility, we examined 53 cone biopsy specimens that were negative for dysplasia or malignancy and their corresponding diagnostic material. Our results can be grouped into 4 general categories.

False-Negative Diagnosis

In 26% of the conization specimens, dysplasia was missed in the initial review of the material and the diagnosis was upgraded to positive (ie, false-negative results). These results are similar to those reported in prior studies. Golbang et al6 reported a false-negative rate of 21%, of which they found half to be the result of interpretive errors. They identified the other half after completely sectioning through the entire block.6 Thompson et al4 reported a false-negative rate of 26%, of which they found half to be the result of interpretative errors and the other half were identified after 3 additional level sections from the blocks. We found that group consensus review helped identify dysplasia in 3 (5.7%) conization specimens, and despite the standard practice at our institution of getting 2 level sections on all cervical conization specimens, an additional level section helped identify dysplasia in 4 (7.5%) conization specimens.

The prior studies did not investigate the use of immunohistochemistry for the tumor suppressor protein p16INK4A, which has become widely acknowledged as a sensitive and specific surrogate marker of HPV-associated high-grade dysplasia.11 Prior reports have indicated that p16 immunohistochemical staining helped identify and reclassify occult dysplastic lesions on biopsies in high-risk HPV-positive women.8 We performed immunohistochemical staining for p16 on the most atypical areas of all the remaining cones and identified dysplasia in 7 (13%) additional conization specimens. The most common cause of difficulty was the differential diagnosis of squamous metaplasia (especially cauterized tissue) or dysplasia. This group of cases highlights the usefulness of additional level sections and p16 immunohistochemistry before rendering a diagnosis of negative for dysplasia or malignancy.

Sampling Error

Follow-up data were available for 20 women with confirmed presurgical diagnosis of HSIL and negative cone diagnoses. Six (30%) of 20 patients were found to have dysplasia or invasive squamous cell carcinoma on subsequent follow-up. This group may represent sampling error. One patient in this group is of particular interest because her follow-up data revealed that she had subsequent ECC and endometrial biopsy specimens with HSIL, and she eventually was diagnosed as having invasive squamous cell carcinoma. This case highlights the possibility that cervical Pap smears and ECCs may contain tissue in the endocervical canal that is higher than in the conization specimen. In addition, the area of dysplasia may be in the vagina or vulva. Overall, the findings emphasize the need for careful follow-up despite a negative conization specimen.

Livasy et al7 reported that 16 (17%) of 93 patients with negative loop electrosurgical excision procedure biopsies had positive follow-up results (carcinoma, HSIL, or LSIL); however, they did not determine whether any of the cones were false negative or whether the diagnosis of presurgical material was correct.7 If we include all of our cases with follow-up from all categories, the overall rate of positive follow-up (carcinoma, HSIL, or LSIL) is 21% (8/39, Table 1).

Overdiagnosis of Presurgical Material

In 28.3% (15/53) of conization specimens, we determined that the prior Pap smear, cervical biopsy specimen, and/or ECC specimen had been overcalled, leading to an unnecessary conization procedure. Of the 39 cases with confirmed negative conization specimens, 8 (20.5%) Pap smears, 10 biopsy specimens, and 1 ECC specimen were downgraded. For 4 cases, multiple specimens were downgraded.

Complications of conventional Pap smears possibly contributed to this category. The majority of the presurgical Pap smears that were overcalled (6 out of 8) were conventional Pap smears. On review of their Pap smears (excluding the false-negative cones), Thompson et al4 found that 10 (45%) of 22 Pap smears were overdiagnosed. Although not stated, all of their specimens were likely conventional slides. Wiegl et al5 identified 22 of 208 cone biopsy specimens with no evidence of dysplasia and attributed the findings to overdiagnosis of conventional Pap smears; they did not address the possibility of false-negative conization specimens or sampling error.

In addition, of the 39 cases with confirmed negative conization specimens, 10 biopsy specimens and 1 ECC specimen were overdiagnosed initially. P16 immunohistochemistry performed on 7 of these biopsy specimens at the time of the study was negative. Thus, the changes were interpreted as squamous metaplasia or LSIL rather than HSIL. Thompson et al4 reviewed their ECC specimens and identified 8 (32%) of 25 cases that were overdiagnosed based on consensus review. Peer review, deeper-level sections, and liberal use of p16 immunohistochemistry at the time of initial evaluation may decrease the number of overcalled presurgical biopsy and/or ECC specimens and thus decrease the number of unnecessary conization procedures performed.

Presurgical Material Confirmed as Dysplasia but No Dysplasia in the Cone or in Subsequent Follow-up Material

No errors were detected in either the presurgical or conization diagnosis in 26.4% of the cones, and the patients’ subsequent follow-up results were negative. The implications of this category are unclear but may result from incomplete tissue examination, regression of the lesion, or possibly complete excision of small foci of HSIL during the biopsy. The actual amount of tissue that is examined microscopically is a small fraction of the tissue resected. Despite at least 3 level sections per block of the cone specimen, in the case of a very small dysplastic focus, it is possible that the lesion was present in the conization specimen but not present in the glass slides. Golbang et al6 identified dysplasia in 11% of their “negative” conization specimens after completely reviewing all levels through the entire block.

In summary, our results, taken as a whole, highlight the usefulness of p16 immunohistochemistry, deeper-level sections, review of prior material, and consensus group review in the pathologic workup of cervical dysplasia. Our findings also emphasize the need for careful follow-up despite a negative conization specimen.

CME/SAM

Upon completion of this activity you will be able to:

  • list possible sources of diagnostic error when a cone biopsy with no dysplasia is encountered.

  • outline additional follow-up measures that may be helpful for the pathologist to order when a cone biopsy with no dysplasia is encountered (ie, tests to be performed and/or review of prior cytology or biopsy material).

  • make recommendations to gynecologists regarding appropriate follow-up of patients with cone biopsies with no dysplasia.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 552. Exam is located at www.ascp.org/ajcpcme.

References

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