- Email: [email protected]
Atypical immature metaplasia (AIM) of the cervix: Is it related to high-grade squamous intraepithelial lesion (HSIL)?
Atypical Immature Metaplasia (AIM) of the Cervix: Is It Related to High-Grade Squamous Intraepithelial Lesion (HSIL)? LI GENG, MD, DENISE C. CONNOLLY, PHD, CHRISTINA ISACSON, MD, BRIGITTE M. RONNETT, MD, AND KATHLEEN R. CHO, MD Atypical immature metaplasia (AIM) is a poorly characterized cervical lesion with uncertain biological and clinical significance. AIM shares some, but not all, morphological features of squamous intraepithelial lesions (SILs). SILs are characterized by human papillomavirus (HPV) positivity and increased cellular proliferation, but these features have not been fully evaluated in AIM. Genomic D N A was extracted from 27 microdissected cervical biopsy specimens diagnosed as AIM. HPV D N A was detected by polymerase chain reaction (PCR), using two different sets of L1 gene consensus primers. HPV types were identified by sequence analysis of PCR products and comparison with published HPV sequences. The cell proliferation index was assessed by inununohistochemicalstaining for Ki-67 (MIB-1) antigen and expressed as the percentage of Ki-67positive cells. Comparison groups included normal cervix (n = 10) and low-grade (LSILs, n = 19) and high-grade sqnanmus intraepithelial lesions (HSILs, n = 11). Intermediate- or high-risk HPV D N A was detected in 67% (18 of 27) of AIM cases. Low-risk HPV DNA was not detected in any of the specimens. The Ki-67 index in AIM (mean, 33.0 + 20.3; median, 29) was comparable to that of LSILs (mean, 21.4 ± 4.6; median, 21) and was significantly higher than that of normal cervix (mean, 11.0 ± 2.1; median, 11) (P < .01) and lower
than that of HSILs (mean, 60.4 ± 13.2; median, 60) (P < .01). Of the cases with available follow-up, I-[PV-positlve AIMs were significantly more likely to have a concurrent or subsequent diagnosis of typical HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 45%) (P = .014). The wide range of Ki-67 indices and variable HPV status in AIM suggest that AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (high-risk HPV-positive, low to moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). HPV testing may be useful in the assessment of atypical epithelial proliferations of the cervix for which a diagnosis of AIM is considered. HUM PATHOL30:345-351. Copyright © 1999 by W.B. Samlders Company Key words: atypical immature metaplasia, dysplasia, squamous intraepithelial lesion, human papillomavirus, Ki-67. Abbreviations: AIM, atypical immature metaplasia; HSIL, highgrade squamous intraepithelial lesion; CIN, cervical intraepithelial neoplasia; SIL, squamous intraepithelia lesion; HPV, human papillomavirus; PCR, polymerase chain reaction; H & E, hematoxylin and eosin.
Atypical immature metaplasia (MM) is a poorly characterized squamous lesion of the cervix with uncertain b i o l o g i c a l a n d c l i n i c a l s i g n i f i c a n c e . A I M is f o u n d
grade, and may be seen in association with SILs. 1,2 However, its etiological relationship, if any, to SIL remains unclear. AIM also may be difficult to distinguish from a variety of reactive/reparative processes within the transformation zone, particularly in the presence of extensive inflammation.I Infection with cancer-associated h u m a n papillomavirus (HPV) types is thought to be necessary for the development of HSILs, and HPV DNA is nearly always detectable using sensitive polymerase chain reaction (PCR)-based methods. In addition, HSILs show markedly increased cellular proliferation as measured by the immunohistochemical detection of Ki-67 (MIB-1).35 In contrast, HPV DNA is infrequently detected in reactive/ reparative epithelia, and cell proliferation may be variable. 6 Cellular proliferation and HPV status have not b e e n fully evaluated in AIM but may provide insights into the relationship of these AIM lesions to bona fide SILs. We d e t e r m i n e d tile HPV status and Ki-67 index (percent Ki-67-positive cells) of 27 cervical lesions diagnosed as AIM. These data were c o m p a r e d with those from a group of well-characterized specimens representing normal cervix, LSIL (CIN1), and HSIL. 3
w i t h i n t h e c e r v i c a l t r a n s f o r m a t i o n z o n e a n d shows i m m a t u r e m e t a p l a s t i c s q u a m o u s cells with less cytological a t y p i a t h a n is s e e n i n typical h i g h - g r a d e s q u a m o u s i n t r a e p i t h e l i a l l e s i o n s (HSILs; also k n o w n as c e r v i c a l i n t r a e p i t h e l i a l n e o p l a s i a 2-3 [ C I N 2 - 3 ] ) . As o r i g i n a l l y d e f i n e d b y C r u m et al, 1 t h e h i s t o l o g i c a l f e a t u r e s o f A I M i n c l u d e (1) a u n i f o r m basal cell p o p u l a t i o n , (2) m i n i real n u c l e a r c r o w d i n g , (3) v a r i a b l e h y p e r c h r o m a t i s m w i t h u n i f o r m c h r o m a t i n d i s t r i b u t i o n i n t h e b a s a l cells, (4) p r e s e r v e d c e l l u l a r polarity, (5) e n l a r g e d o r m u l t i p l e n u c l e i c o n f i n e d to s u p r a b a s a l areas, a n d (6) a b s e n c e o f a b n o r m a l m i t o t i c figures. A I M s h a r e s s o m e m o r p h o l o g i cal a n d p a t h o g e n e t i c f e a t u r e s w i t h s q u a m o u s i n t r a e p i t h e l i a l l e s i o n s (SILs, also k n o w n as c e r v i c a l i n t r a e p i t h e lial n e o p l a s i a [ C I N ] ) , p a r t i c u l a r l y t h o s e t h a t a r e h i g h
From the Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD; and the Department of Pathology, The NewYork Hospital-Cornell Medical Center, NewYork, NY. Accepted for publication November 13, 1998. Supported by funds from the Richard W. TeLinde endowment, Johns Hopkins University School of Medicine and the Caring Collection, Annapolis, MD. Address correspondence and reprint requests to Kathleen R. Cho, MD, Department of Pathology, The University of Michigan Medical School, 4301 MSRBIII, Box 0638, 1150 West Medical Center Dr, Ann Arbor, M1 48109. Copyright © 1999 by W.B. Saunters Company 0046-8177/99/3003-0016510.00/0
345
MATERIALS AND METHODS
Specimens All specimens were o b t a i n e d from the Surgical Pathology archives of The Johns Hopkins a n d Cornell-New York Hospitals a n d retrieved o n the basis o£ the original diagnosis of
HUMAN PATHOLOGY
Volume 30, No, 3 (March 1999)
atypical immature metaplasia or immature metaplasia with atypia. Hematoxylin and eosin (H & E)-stained sections from each specimen were reviewed by two pathologists (K.R.C. and B.M.R.), and the case was included in the study if both pathologists concurred with the original AIM diagnosis. A total of 27 cervical biopsy specimens diagnosed as AIM were ultimately collected for analysis. A set of previonsly analyzed cervical biopsy specimens including normal cervix (n = 10), LSILs (n = 19), and HSILs (n = 11) were used as comparison groups. The normal cervix specimens contained regions of mature squamous metaplasia. HPV status and Ki-67 immunostaining results of the normal cervix, LSIL, and HSIL specimens have been reported previously.~ HPV D e t e c t i o n a n d T y p i n g
Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue sections of each specimen diagnosed as AIM using standard methods as we have previously described, 7 Lesion-containing regions were microdissected from 5-pro sections with 22-gauge needles under a light microscope, using adjacent H&E-stained sections as dissection guides. HPV DNA was detected by PCR amplification using two different sets of L1 consensus primers, L1C1/L1C2 s and MY09/MY11. 9 In some cases, PCR using E6 primers specific for HPV types 16, 18, 33, or 58 also was performed. Each group of L1 consensus region PCR amplifications included a positive control (CaSki cell line DNA [HPV16 positive]) as well as a negative (no DNA) control. Adequate DNA quality was verified by amplification of a 260-bp segment of the [3-globin gene under identical PCR conditions. An AIM lesion was considered HPV positive if the typing with both sets of L1 consensus primers was concordant, or if typing by one set of L1 consensus primers was corroborated by detection of type-specific HPV E6 DNA. Ki-67 A n t i g e n L a b e l i n g a n d C a l c u l a t i o n o f t h e Ki-67 I n d e x
lesion being evaluated. We generally found the Ki-67 labeling pattern to be quite uniform within each lesion studied. For cases with slight variability of staining within a given lesion, the region of highest activity was selected for evaluation. Only cells within the grid squares were counted, and the field was shifted randomly to sample 250 to 1,000 cells. A positively labeled nucleus was defined as any nucleus with detectable staining above the negative control background level. The Ki-67 index was expressed as the number of positively labeled cells per 100 epithelial cells (% positive). Low, moderate, and high Ki-67 labeling indices were defined as 15 or less, 16 to 33, and 34 or greater, respectively. The "low" and "high" cutoffs were chosen to reflect the mean labeling index of normal cervix plus two standard deviations and mean labeling index of HSIL minus two standard deviations. Follow-Up
The Cytopathology and Surgical Pathology files at the Johns Hopkins and New York Hospitals were searched for cervical cytology or tissue specimens obtained at the same time or after the index AIM biopsy specimen. Follow-up was considered "not available" if there were no subsequent cytological (Pap smear) or tissue specimens. Those patients whose only additional material included a negative Pap smear concurrent with the AIM diagnosis were also considered to have no available follow-up. Statistical A n a l y s i s
The Kruskal-Wallis test was used to test for significance in pairwise comparisons of the Ki-67 index in AIM versus normal cervix, LSILs, and HSILs. The Fisher's exact test (two-tailed) was used for statistical analysis of follow-up data.
RESULTS
Five-micron sections from each specimen were immunohistochemically stained for MIB-1 antigen using the mouse monoclonal antibody Ki-67 (Dako, Carpenteria, CA). Immunostaining was p e r f o r m e d on a Tech Mate I000 automated stainer (BioTek Solutions, Inc, Santa Barbara, CA) per the manufacturer's protocol. Antigen enhancement was performed by immersing the slides in a sodium citrate buffer and heating in a steamer for 20 minutes. A conjugated reporter enzyme (peroxidase) was used for detection of a chromogenic substrate, and tissues were lightly connterstained with hematoxylin. The number of positively labeled nuclei in at least 250 cells of the full-thickness epithelium in the normal and lesional tissues was determined. Cell scoring was assisted by positioning a 10 × 10 square grid in the microscopic eyepiece to encompass the full thickness of the epithelium within the
H i s t o p a t h o l o g i c A s s e s s m e n t o f A I M Lesions
All b i o p s y s p e c i m e n s d i a g n o s e d as A I M were c h a r a c t e r i z e d by a l a c k o f m a t u r a t i o n in t h e m e t a p l a s t i c s q u a m o u s e p i t h e l i u m . I n s o m e cases, m a t u r a t i o n was e v i d e n t in t h e m o s t s u p e r f i c i a l layers, b u t in o t h e r s t h e r e was n o n e . T h e n u c l e a r c h a n g e s w e r e v a r i a b l e f r o m case to case, with s o m e s h o w i n g m i l d h y p e r c h r o m a sia w i t h o u t a d d i t i o n a l c h r o m a t i n i r r e g u l a r i t i e s a n d o t h e r s s h o w i n g m o r e p r o m i n e n t h y p e r c h r o m a s i a in c o n j u n c t i o n with m i l d c h r o m a t i n i r r e g u l a r i t i e s . T h e h y p e r c h r o m a s i a was g e n e r a l l y " s m o o t h " a n d d e n s e , w i t h o u t t h e coarse, i r r e g u l a r c h r o m a t i n c l u m p i n g t h a t is s e e n in typical HSIL. F o r t h o s e cases with g r e a t e r
FIGURE I. Representative AIM lesions. Case 2: Atypical immature metaplc~sia. (A) Lesion shows loss of maturation and scattered enlarged, hyperchromatic nuclei. Mitotic figures were not identified above the parabasal layer. (H&E.) (B) Immunoperoxidase stain for Ki-67 shows numerous positive nuclei in the lower two thirds of the epithelium, with scattered positive nuclei in the upper third, Ki-67 proliferation index is 25%. HPV PCR was negative. Follow-up was negative for intraepithelial lesions. Case 13: Atypical immature metaplasia. (C) Lesion is characterized by thickened squamous epithelium, a widened parabasal zone, and some loss of maturation. Cytological atypia is minimal, and mitotic figures are not evident. (H&E,) (D) Ki-67 proliferation index is 43%, with numerous positive nuclei throughout all layers of the epithelium. PCR was positive for HPV 33, HSILwas identified on follow-up. Case 25: Atypical immature metaplasia versus high-grade squamous intraepithelial lesion. (E) Squamous epithelium shows loss of maturation with enlarged, hyperchromatic nuclei but no evident mitotic activity. (H&E.) (F) Ki-67 proliferation index is markedly increased (71%), with positive nuclei throughout all layers of the epithelium. PCR was positive for HPV 16, HSILwas identified on follow-up. Case 22: Atypical immature metaplasia versus high-grade squamous intraepithelial lesion. (G) Lesion is characterized by loss of maturation and enlarged, hyperchromatic nuclei, but the epithelium is thin, and there are no evident mitotic figures. (H&E.) (H) Ki-67 index is 47%, with positive nuclei at all levels of the epithelium. PCR was positive for HPVs 31 and 18, Follow-up was not available.
346
AIM OF THE CERVIX (Geng et al)
347
HUMAN PATHOLOGY
Volume 30, No. 3 (March 1999)
nuclear hyperchromasia, a diagnosis of HSIL was seriously considered but was not rendered because of a paucity of mitotic figures (usually none were identified) and lack of coarsely clumped chromatin and nuclear membrane irregularities. Many of the cases, especially those with greater cytological atypia, had been reviewed at a consensus conference as part of the routine quality
assurance for gynecologic specimens, and the consensus opinion was that a definitive diagnosis of HSIL could not be made but was clearly in the differential diagnosis. Representative examples of AIM lesions are shown in Figure 1 (panels A, C, E, and G) and for comparison purposes, typical examples of normal cervix, LSIL, and HSIL are shown in Figure 2 (panels A, C, and E).
FIGURE 2. (A) Normal cervix. (H&E.) (B) Normal cervix. Ki-67 immunostain shows immunoreactive nuclei only in the parabasal zone. (C) LSIL.(H&E.) Basal zone is widened, and upper two thirds of epithelium contains cells that have enlarged, hyperchromatic nuclei and cytoplasmic clearing (koilocytosis). (D) LSIL.Ki-67 immunostain shows numerous immunoreactive nuclei throughout the basal zone and scattered immunoreactive nuclei in the upper two thirds of the epithelium, reflecting an elevated proliferation index. (E) HSIL. (H&E.) Squamous epithelium shows loss of maturation, cells with enlarged, hyperchromatic nuclei, and several mitotic figures, including one in the upper third of the epithelium. (F) HSIL. Ki-67 immunostain shows numerous immunoreactive nuclei throughout the entire epithelium, indicative of a markedly elevated proliferation index.
348
AIM OF THE CERVIX (Geng et al) HPV D e t e c t i o n a n d Typing
9O
The normal cervical biopsy specimens (n = 10) selected for comparison with the AIM lesions were all HPV negative, and the LSILs (n = 19) and HSILs (n = 11) were HPV positive, s T h e results of HPV detection and typing in AIM are summarized in Table 1. Intermediate or high-risk HPV DNA was detected in 18 of 27 AIM lesions (67%) by PCR. Low-risk HPV DNA was not detected in any AIM lesion. Eight of the 18 (44%) HPV-positive AIM specimens contained high-risk HPV types 16 or 18. O f these eight, three were coinfected with intermediate-risk types o f H P V (HPV types 31 or 33). Ten of 18 (56%) HPV-positive AIM lesions contained exclusively intermediate-risk HPVs (types 31, 33, 35, or 58). Nine of 27 (33%) AIM lesions were HPV-negative using both sets of HPV consensus PCR primers.
80
~:
60
:
-.oo
i o 0
FIGURE 3.
TABLE 1. Summary of HPV Typing, Ki-67 Indices, and Follow-up in AIM Lesions
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Negative Negative Negative Negative Negative Negative Negative Negative Negative 58 58 31 33 33 33 33 33 35 35 33/18 31/18 31/18 16 16 16 16 16
9 25 2 65 43 46 16 14 9 7 21 30 43 68 45 29 12 38 16 15 59 47 28 57 71 49 26
•
:
t
I
!
Normal Cervix
r
i
1
LSIL
HSIL
!r.:
AIM
HPV HPV Negative Positive AIM AIM
Ki-67 indices in normal cervix, SILs, a n d AIM. --,
median.
We and others have previously shown that the Ki-67 labeling index increases with increasing lesion grade. 35,10 The mean Ki-67 labeling indices (+standard deviation) of normal cervix, LSILs, and HSILs, as determined by our previous analysis, were 11.0 + 2.1 (median, 11), 21.4 + 4.6 (median, 21), and 60.4 + 13.2 (median, 60), respectively. S T h e Ki-67 indices of the individual AIM lesions are shown in Table 1 and Figure 3. Overall, the Ki-67 index o f AIM (mean, 33.0 + 20.3; median, 29) was slightly higher than, but comparable to, that of LSILs, and significantly higher than that of normal cervix ( P < .01), and significantly lower than that of
Ki-67Index (%)
, !
40
Ki-67 I n d e x
HPV Status/Type
.
Follow-up NA Negative NA NA HSIL Negative Negative Negative Negative HSIL HSIL HSIL HSIL
HSILs (P < .01). However, the range of Ki-67 indices in AIM clearly overlapped those of normal cervix, LSIL, and HSIL (Fig 3). The mean Ki-67 index of HPVpositive AIM (mean, 36.7-+ 19.4; median, 34) was h i g h e r than that of HPV-negative AIM (mean, 25.4 + 21.2; median, 16), but this difference did not achieve statistical significance ( P = .117). The Ki-67 index of HPV-positive AIM was significantly higher than that of normal cervix ( P < .01) and LSILs ( P < .05), and significantly lower than that of HSILs ( P < .01). The Ki-67 index of HPV-negative AIM did not differ significantly from normal cervix ( P > .05) or LSILs
(P > .05). In normal cervix, Ki-67 immunopositivity was confined to nuclei in the parabasal region (Fig 2A, B). In LSILs, the Ki-67-positive nuclei were distributed t h r o u g h o u t the lower two-thirds of the epithelium, with relative sparing of the nuclei in the u p p e r third (Fig 2C, D). The HSILs typically showed diffuse Ki-67 immunostaining of nuclei at all levels of the epithelium (Fig 2E, F). In the AIM specimens, the distribution of Ki-67positive nuclei was quite variable. Several examples of HPV-positive and HPV-negative AIM lesions are shown in Figure 1B, 1D, and 1E In some cases, the distribution o f Ki-67-positive cells resembled the pattern seen in normal cervix, whereas others displayed staining distributions similar to those seen in LSILs or HSILs. Follow-Up
NA HSIL HSIL Negative HSIL Negative HSIL
C o n c u r r e n t or subsequent cytological or tissue specimens were available from 21 of the 27 patients diagnosed with AIM. C o n c u r r e n t or subsequent HSIL was diagnosed in 13 of the 21 patients. In all cases, HSIL was diagnosed concurrently or within 3 months of the AIM diagnosis. Twelve of the 13 HSILs were identified in cervical biopsy specimens, and one was identified in a cytological specimen (Pap smear). HPV-positive AIMs were significantly more likely to have a c o n c u r r e n t or subsequent diagnosis of HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 16.7%) (P = .014 by two-tailed Fisher's exact test).
NA NA Squamous carcinoma HSIL HSIL HSIL Negative
Abbreviation: NA, not available.
349
HUMAN PATHOLOGY
Volume 30, No. 3 (March 1999)
DISCUSSION T h e wide range of Ki-67 indices and variable HPV status in AIM suggest that the histopathologic spectrum of lesions that encompass the entity we currently regard as AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (highrisk HPV-positive, low-moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). The HPV-positive AIM lesions were shown to contain the same HPV types (high- and intermediaterisk HPVs) typically identified in HSILs. Interestingly, more than half of the AIM lesions in our group contained intermediate-risk HPV types. Most HPVpositive AIMs were shown to have c o n c u r r e n t or subseq u e n t HSIL within a 3-month period. These findings support the use of HPV testing in the assessment of atypical epithelial proliferations of the cervix in which a diagnosis of AIM is considered. Given our Ki-67 immunostaining results in AIM, we would now interpret the more atypical lesions (ie, those with greater nuclear atypia and elevated Ki-67 indices) as HSIL outright, despite the paucity of mitotic figures. Notably, of 14 AIM lesions for which an HSIL diagnosis was strongly considered, 12 were HPV positive. It is also reasonable to make a diagnosis of AIM versus HSIL (or AIM versus HSIL, favor HSIL) for these more atypical AIM lesions, with the understanding that such cases should probably be managed as HSIL. Determining the appropriate m a n a g e m e n t of HPVnegative AIM lesions is significantly more problematic. We were unable to d o c u m e n t the development of HSIL in most patients with HPV-negative AIM, suggesting that a subset of AIM likely represents benign reactive conditions. All of the HPV-negative AIM lesions with low Ki-67 indices had negative follow-up. However, c o n c u r r e n t HSIL was identified in one patient with HPV-negative AIM. This particular AIM lesion showed significant elevation of the Ki-67 index. Although it is possible that HPV was not detected by PCR-based methods in this case, we think this is unlikely because in situ hybridization for detection of HPV nucleic acids was also negative (Dr Srihari Kadkhol, personal communication). Given that at least a few HPV-negative AIM lesions may be associated with HSIL, additional tissue studies and close clinical follow-up of patients with such lesions would seem prudent--particularly for those with lesions showing elevated cellular proliferation. For those pathologists without routine access to HPV typing and Ki-67 immunostaining, an alternative approach to diagnosis may be considered. We suggest a diagnosis of AIM, followed by a comment. For those AIM lesions that are highly suspicious for, but not diagnostic of, HSIL, the c o m m e n t should specify that although a benign reactive process is in the differential diagnosis, the lesion may represent an HSIL or HSIL precursor, because similar lesions, particularly when shown to be HPV positive, are strongly associated with either c o n c u r r e n t or subsequent development of more
350
overt HSILs. For those lesions that are less suspicious, the c o m m e n t should state that although HSIL is in the differential diagnosis, a benign reactive process is favored. Not surprisingly, the distinction between AIM and HSIL is often difficult and raises the possibility that the two entities may be related, at least in some cases. Although mild, moderate, and severe dysplasias of the cervix (ie, LSILs and HSILs) have been previously thought to represent a morphological and biological c o n t i n u u m in the progression of cervical cancer, more recent analyses of the natural history of cervical dysplasias suggest the possibility that LSILs and HSILs might represent distinct entities with different potential for progression to cancer (reviewed by Kiviat et apl). Therefore, not all HSILs necessarily arise from preexisting LSILs, and there may be other morphologically recognizable lesions that serve as HSIL a n t e c e d e n t s / precursors. Furthermore, in gynecologic cytology, the diagnosis of atypical squamous cells of u n d e t e r m i n e d significance composed of immature metaplastic cells is associated with a greater risk of progression to, or association with, HSILs than those atypical squamous cells of u n d e t e r m i n e d significance cases composed of mature cells. 12 We suggest the possibility that the HPVpositive AIM lesions with low to moderate elevations of the Ki-67 index might represent HSIL precursors. Clearly, larger studies o f AIM are required to confirm or refute such a hypothesis. In summary, our findings suggest that AIM, at least as recognized by pathologists at our institutions, likely represents a heterogeneous group o f lesions. Those that are positive for high- or intermediate-risk HPV types probably represent HSILs or HSIL precursors, and those that are HPV negative may represent benign reactive conditions. The strong association of concurrent or subsequent HSIL with AIM, particularly those that are HPV positive, suggests n e e d for additional tissue studies and close clinical follow-up of patients with these lesions.
A&nowledgment. The authors gratefully acknowledge the expert technical assistance of Wes Gage and George Pettis in the Ki-67 immunostaining, and Drs Robert J. Kurman and Mark Sherman for helpful discussions and critical review of the manuscript. REFERENCES 1. Crum CP, Egawa K, FuY-S, et al: Atypical immature metaplasia (AIM): A subset of human papilloma virus infection of the cervix. Cancer 51:2214-2219, 1983 2. Ward BE, Saleh AM, Williams JV, et al: Atypical immature metaplasia of the cervix: A reevaluation. Lab Invest 64:42A, 1997 (abstr) 3. Isacson C, Kessis TD, Hedrick L, et al: Both cell proliferation and apoptosis increase with lesion grade in cervical neoplasia but do not correlate with human papillomavirus type. Cancer Res 56:669674, 1996 4. Dunton CJ, van Hoeven KH, Kovatich MS, et al: Ki-67 antigen staining as an adjunct to identifying cervical intraepithelial neoplasia. Gynecol Onco164:451-455, 1997
AIM OF THE CERVIX (Geng et al) 5. A1-Saleh W, Delvenne P, Greimers R, et al: Assessment of Ki-67 antigen immunostaining in squamous intraepithelial lesions of the uterine cervix. A m J Clin Pathol 104:154-160, 1995 6. van Hoeven KH, Ramondetta L, Kovatich AJ, et al: Quantitative image analysis of MIB-1 reactivity in inflammatory, hyperplastic, and neoplastic endocervical lesions. Int J Gynecol Pathol 16:15-21, 1997 7. Kessis TD, Silberman MA, Sherman M, et al: Rapid identification of patient specimens with microsatellite DNA markers. Mod Pathol 9:183-188, 1996 8. Yoshikawa H, Kawana T, Kitagawa K, et al: Detection and typing of multiple genital human papillomaviruses by DNA amplification with consensus primers.JpnJ Cancer Res 82:524-531, 1991 9. Manos M, Ting Y, Wright A, et al: The use ofpolymerase chain reaction amplification for the detection of genital human papillomaviruses, in Furth M, Greaves M (eds): Molecular Diagnostics of
Human Cancer: Cancer Cells. Cold Spring Harbor, NY, Cold Spring Laboratory, 1989, pp 209-214 10. Payne S, Kernohan NM, Walker F: Proliferation in the normal cervix and in preinvasive cervical lesions. J Clin Pathol 49:66%671, 1996 11. Kiviat NB, Critchlow CW, Kurman RJ: Reassessment of the morphological continuum of cervical intraepithelial lesions: Does it reflect different stages in the progression to cervical carcinoma?, in Mufioz N, Bosch FX, Shah KV, Meheus A (eds): The Epidemiology of Cervical Cancer and Human Papillomavirus. Lyon, France, International Agency for Research on Cancer, 1992, pp 59-66 12. Sheils LA, Wilbur DC: Atypical squamous cells of undetermined significance: Stratification of the risk of association with, or progression to, squamous intraepithelial lesions based on morphologic subcategorization. Acta Qftol 41:1065-1072, 1997
351
than that of HSILs (mean, 60.4 ± 13.2; median, 60) (P < .01). Of the cases with available follow-up, I-[PV-positlve AIMs were significantly more likely to have a concurrent or subsequent diagnosis of typical HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 45%) (P = .014). The wide range of Ki-67 indices and variable HPV status in AIM suggest that AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (high-risk HPV-positive, low to moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). HPV testing may be useful in the assessment of atypical epithelial proliferations of the cervix for which a diagnosis of AIM is considered. HUM PATHOL30:345-351. Copyright © 1999 by W.B. Samlders Company Key words: atypical immature metaplasia, dysplasia, squamous intraepithelial lesion, human papillomavirus, Ki-67. Abbreviations: AIM, atypical immature metaplasia; HSIL, highgrade squamous intraepithelial lesion; CIN, cervical intraepithelial neoplasia; SIL, squamous intraepithelia lesion; HPV, human papillomavirus; PCR, polymerase chain reaction; H & E, hematoxylin and eosin.
Atypical immature metaplasia (MM) is a poorly characterized squamous lesion of the cervix with uncertain b i o l o g i c a l a n d c l i n i c a l s i g n i f i c a n c e . A I M is f o u n d
grade, and may be seen in association with SILs. 1,2 However, its etiological relationship, if any, to SIL remains unclear. AIM also may be difficult to distinguish from a variety of reactive/reparative processes within the transformation zone, particularly in the presence of extensive inflammation.I Infection with cancer-associated h u m a n papillomavirus (HPV) types is thought to be necessary for the development of HSILs, and HPV DNA is nearly always detectable using sensitive polymerase chain reaction (PCR)-based methods. In addition, HSILs show markedly increased cellular proliferation as measured by the immunohistochemical detection of Ki-67 (MIB-1).35 In contrast, HPV DNA is infrequently detected in reactive/ reparative epithelia, and cell proliferation may be variable. 6 Cellular proliferation and HPV status have not b e e n fully evaluated in AIM but may provide insights into the relationship of these AIM lesions to bona fide SILs. We d e t e r m i n e d tile HPV status and Ki-67 index (percent Ki-67-positive cells) of 27 cervical lesions diagnosed as AIM. These data were c o m p a r e d with those from a group of well-characterized specimens representing normal cervix, LSIL (CIN1), and HSIL. 3
w i t h i n t h e c e r v i c a l t r a n s f o r m a t i o n z o n e a n d shows i m m a t u r e m e t a p l a s t i c s q u a m o u s cells with less cytological a t y p i a t h a n is s e e n i n typical h i g h - g r a d e s q u a m o u s i n t r a e p i t h e l i a l l e s i o n s (HSILs; also k n o w n as c e r v i c a l i n t r a e p i t h e l i a l n e o p l a s i a 2-3 [ C I N 2 - 3 ] ) . As o r i g i n a l l y d e f i n e d b y C r u m et al, 1 t h e h i s t o l o g i c a l f e a t u r e s o f A I M i n c l u d e (1) a u n i f o r m basal cell p o p u l a t i o n , (2) m i n i real n u c l e a r c r o w d i n g , (3) v a r i a b l e h y p e r c h r o m a t i s m w i t h u n i f o r m c h r o m a t i n d i s t r i b u t i o n i n t h e b a s a l cells, (4) p r e s e r v e d c e l l u l a r polarity, (5) e n l a r g e d o r m u l t i p l e n u c l e i c o n f i n e d to s u p r a b a s a l areas, a n d (6) a b s e n c e o f a b n o r m a l m i t o t i c figures. A I M s h a r e s s o m e m o r p h o l o g i cal a n d p a t h o g e n e t i c f e a t u r e s w i t h s q u a m o u s i n t r a e p i t h e l i a l l e s i o n s (SILs, also k n o w n as c e r v i c a l i n t r a e p i t h e lial n e o p l a s i a [ C I N ] ) , p a r t i c u l a r l y t h o s e t h a t a r e h i g h
From the Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD; and the Department of Pathology, The NewYork Hospital-Cornell Medical Center, NewYork, NY. Accepted for publication November 13, 1998. Supported by funds from the Richard W. TeLinde endowment, Johns Hopkins University School of Medicine and the Caring Collection, Annapolis, MD. Address correspondence and reprint requests to Kathleen R. Cho, MD, Department of Pathology, The University of Michigan Medical School, 4301 MSRBIII, Box 0638, 1150 West Medical Center Dr, Ann Arbor, M1 48109. Copyright © 1999 by W.B. Saunters Company 0046-8177/99/3003-0016510.00/0
345
MATERIALS AND METHODS
Specimens All specimens were o b t a i n e d from the Surgical Pathology archives of The Johns Hopkins a n d Cornell-New York Hospitals a n d retrieved o n the basis o£ the original diagnosis of
HUMAN PATHOLOGY
Volume 30, No, 3 (March 1999)
atypical immature metaplasia or immature metaplasia with atypia. Hematoxylin and eosin (H & E)-stained sections from each specimen were reviewed by two pathologists (K.R.C. and B.M.R.), and the case was included in the study if both pathologists concurred with the original AIM diagnosis. A total of 27 cervical biopsy specimens diagnosed as AIM were ultimately collected for analysis. A set of previonsly analyzed cervical biopsy specimens including normal cervix (n = 10), LSILs (n = 19), and HSILs (n = 11) were used as comparison groups. The normal cervix specimens contained regions of mature squamous metaplasia. HPV status and Ki-67 immunostaining results of the normal cervix, LSIL, and HSIL specimens have been reported previously.~ HPV D e t e c t i o n a n d T y p i n g
Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue sections of each specimen diagnosed as AIM using standard methods as we have previously described, 7 Lesion-containing regions were microdissected from 5-pro sections with 22-gauge needles under a light microscope, using adjacent H&E-stained sections as dissection guides. HPV DNA was detected by PCR amplification using two different sets of L1 consensus primers, L1C1/L1C2 s and MY09/MY11. 9 In some cases, PCR using E6 primers specific for HPV types 16, 18, 33, or 58 also was performed. Each group of L1 consensus region PCR amplifications included a positive control (CaSki cell line DNA [HPV16 positive]) as well as a negative (no DNA) control. Adequate DNA quality was verified by amplification of a 260-bp segment of the [3-globin gene under identical PCR conditions. An AIM lesion was considered HPV positive if the typing with both sets of L1 consensus primers was concordant, or if typing by one set of L1 consensus primers was corroborated by detection of type-specific HPV E6 DNA. Ki-67 A n t i g e n L a b e l i n g a n d C a l c u l a t i o n o f t h e Ki-67 I n d e x
lesion being evaluated. We generally found the Ki-67 labeling pattern to be quite uniform within each lesion studied. For cases with slight variability of staining within a given lesion, the region of highest activity was selected for evaluation. Only cells within the grid squares were counted, and the field was shifted randomly to sample 250 to 1,000 cells. A positively labeled nucleus was defined as any nucleus with detectable staining above the negative control background level. The Ki-67 index was expressed as the number of positively labeled cells per 100 epithelial cells (% positive). Low, moderate, and high Ki-67 labeling indices were defined as 15 or less, 16 to 33, and 34 or greater, respectively. The "low" and "high" cutoffs were chosen to reflect the mean labeling index of normal cervix plus two standard deviations and mean labeling index of HSIL minus two standard deviations. Follow-Up
The Cytopathology and Surgical Pathology files at the Johns Hopkins and New York Hospitals were searched for cervical cytology or tissue specimens obtained at the same time or after the index AIM biopsy specimen. Follow-up was considered "not available" if there were no subsequent cytological (Pap smear) or tissue specimens. Those patients whose only additional material included a negative Pap smear concurrent with the AIM diagnosis were also considered to have no available follow-up. Statistical A n a l y s i s
The Kruskal-Wallis test was used to test for significance in pairwise comparisons of the Ki-67 index in AIM versus normal cervix, LSILs, and HSILs. The Fisher's exact test (two-tailed) was used for statistical analysis of follow-up data.
RESULTS
Five-micron sections from each specimen were immunohistochemically stained for MIB-1 antigen using the mouse monoclonal antibody Ki-67 (Dako, Carpenteria, CA). Immunostaining was p e r f o r m e d on a Tech Mate I000 automated stainer (BioTek Solutions, Inc, Santa Barbara, CA) per the manufacturer's protocol. Antigen enhancement was performed by immersing the slides in a sodium citrate buffer and heating in a steamer for 20 minutes. A conjugated reporter enzyme (peroxidase) was used for detection of a chromogenic substrate, and tissues were lightly connterstained with hematoxylin. The number of positively labeled nuclei in at least 250 cells of the full-thickness epithelium in the normal and lesional tissues was determined. Cell scoring was assisted by positioning a 10 × 10 square grid in the microscopic eyepiece to encompass the full thickness of the epithelium within the
H i s t o p a t h o l o g i c A s s e s s m e n t o f A I M Lesions
All b i o p s y s p e c i m e n s d i a g n o s e d as A I M were c h a r a c t e r i z e d by a l a c k o f m a t u r a t i o n in t h e m e t a p l a s t i c s q u a m o u s e p i t h e l i u m . I n s o m e cases, m a t u r a t i o n was e v i d e n t in t h e m o s t s u p e r f i c i a l layers, b u t in o t h e r s t h e r e was n o n e . T h e n u c l e a r c h a n g e s w e r e v a r i a b l e f r o m case to case, with s o m e s h o w i n g m i l d h y p e r c h r o m a sia w i t h o u t a d d i t i o n a l c h r o m a t i n i r r e g u l a r i t i e s a n d o t h e r s s h o w i n g m o r e p r o m i n e n t h y p e r c h r o m a s i a in c o n j u n c t i o n with m i l d c h r o m a t i n i r r e g u l a r i t i e s . T h e h y p e r c h r o m a s i a was g e n e r a l l y " s m o o t h " a n d d e n s e , w i t h o u t t h e coarse, i r r e g u l a r c h r o m a t i n c l u m p i n g t h a t is s e e n in typical HSIL. F o r t h o s e cases with g r e a t e r
FIGURE I. Representative AIM lesions. Case 2: Atypical immature metaplc~sia. (A) Lesion shows loss of maturation and scattered enlarged, hyperchromatic nuclei. Mitotic figures were not identified above the parabasal layer. (H&E.) (B) Immunoperoxidase stain for Ki-67 shows numerous positive nuclei in the lower two thirds of the epithelium, with scattered positive nuclei in the upper third, Ki-67 proliferation index is 25%. HPV PCR was negative. Follow-up was negative for intraepithelial lesions. Case 13: Atypical immature metaplasia. (C) Lesion is characterized by thickened squamous epithelium, a widened parabasal zone, and some loss of maturation. Cytological atypia is minimal, and mitotic figures are not evident. (H&E,) (D) Ki-67 proliferation index is 43%, with numerous positive nuclei throughout all layers of the epithelium. PCR was positive for HPV 33, HSILwas identified on follow-up. Case 25: Atypical immature metaplasia versus high-grade squamous intraepithelial lesion. (E) Squamous epithelium shows loss of maturation with enlarged, hyperchromatic nuclei but no evident mitotic activity. (H&E.) (F) Ki-67 proliferation index is markedly increased (71%), with positive nuclei throughout all layers of the epithelium. PCR was positive for HPV 16, HSILwas identified on follow-up. Case 22: Atypical immature metaplasia versus high-grade squamous intraepithelial lesion. (G) Lesion is characterized by loss of maturation and enlarged, hyperchromatic nuclei, but the epithelium is thin, and there are no evident mitotic figures. (H&E.) (H) Ki-67 index is 47%, with positive nuclei at all levels of the epithelium. PCR was positive for HPVs 31 and 18, Follow-up was not available.
346
AIM OF THE CERVIX (Geng et al)
347
HUMAN PATHOLOGY
Volume 30, No. 3 (March 1999)
nuclear hyperchromasia, a diagnosis of HSIL was seriously considered but was not rendered because of a paucity of mitotic figures (usually none were identified) and lack of coarsely clumped chromatin and nuclear membrane irregularities. Many of the cases, especially those with greater cytological atypia, had been reviewed at a consensus conference as part of the routine quality
assurance for gynecologic specimens, and the consensus opinion was that a definitive diagnosis of HSIL could not be made but was clearly in the differential diagnosis. Representative examples of AIM lesions are shown in Figure 1 (panels A, C, E, and G) and for comparison purposes, typical examples of normal cervix, LSIL, and HSIL are shown in Figure 2 (panels A, C, and E).
FIGURE 2. (A) Normal cervix. (H&E.) (B) Normal cervix. Ki-67 immunostain shows immunoreactive nuclei only in the parabasal zone. (C) LSIL.(H&E.) Basal zone is widened, and upper two thirds of epithelium contains cells that have enlarged, hyperchromatic nuclei and cytoplasmic clearing (koilocytosis). (D) LSIL.Ki-67 immunostain shows numerous immunoreactive nuclei throughout the basal zone and scattered immunoreactive nuclei in the upper two thirds of the epithelium, reflecting an elevated proliferation index. (E) HSIL. (H&E.) Squamous epithelium shows loss of maturation, cells with enlarged, hyperchromatic nuclei, and several mitotic figures, including one in the upper third of the epithelium. (F) HSIL. Ki-67 immunostain shows numerous immunoreactive nuclei throughout the entire epithelium, indicative of a markedly elevated proliferation index.
348
AIM OF THE CERVIX (Geng et al) HPV D e t e c t i o n a n d Typing
9O
The normal cervical biopsy specimens (n = 10) selected for comparison with the AIM lesions were all HPV negative, and the LSILs (n = 19) and HSILs (n = 11) were HPV positive, s T h e results of HPV detection and typing in AIM are summarized in Table 1. Intermediate or high-risk HPV DNA was detected in 18 of 27 AIM lesions (67%) by PCR. Low-risk HPV DNA was not detected in any AIM lesion. Eight of the 18 (44%) HPV-positive AIM specimens contained high-risk HPV types 16 or 18. O f these eight, three were coinfected with intermediate-risk types o f H P V (HPV types 31 or 33). Ten of 18 (56%) HPV-positive AIM lesions contained exclusively intermediate-risk HPVs (types 31, 33, 35, or 58). Nine of 27 (33%) AIM lesions were HPV-negative using both sets of HPV consensus PCR primers.
80
~:
60
:
-.oo
i o 0
FIGURE 3.
TABLE 1. Summary of HPV Typing, Ki-67 Indices, and Follow-up in AIM Lesions
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Negative Negative Negative Negative Negative Negative Negative Negative Negative 58 58 31 33 33 33 33 33 35 35 33/18 31/18 31/18 16 16 16 16 16
9 25 2 65 43 46 16 14 9 7 21 30 43 68 45 29 12 38 16 15 59 47 28 57 71 49 26
•
:
t
I
!
Normal Cervix
r
i
1
LSIL
HSIL
!r.:
AIM
HPV HPV Negative Positive AIM AIM
Ki-67 indices in normal cervix, SILs, a n d AIM. --,
median.
We and others have previously shown that the Ki-67 labeling index increases with increasing lesion grade. 35,10 The mean Ki-67 labeling indices (+standard deviation) of normal cervix, LSILs, and HSILs, as determined by our previous analysis, were 11.0 + 2.1 (median, 11), 21.4 + 4.6 (median, 21), and 60.4 + 13.2 (median, 60), respectively. S T h e Ki-67 indices of the individual AIM lesions are shown in Table 1 and Figure 3. Overall, the Ki-67 index o f AIM (mean, 33.0 + 20.3; median, 29) was slightly higher than, but comparable to, that of LSILs, and significantly higher than that of normal cervix ( P < .01), and significantly lower than that of
Ki-67Index (%)
, !
40
Ki-67 I n d e x
HPV Status/Type
.
Follow-up NA Negative NA NA HSIL Negative Negative Negative Negative HSIL HSIL HSIL HSIL
HSILs (P < .01). However, the range of Ki-67 indices in AIM clearly overlapped those of normal cervix, LSIL, and HSIL (Fig 3). The mean Ki-67 index of HPVpositive AIM (mean, 36.7-+ 19.4; median, 34) was h i g h e r than that of HPV-negative AIM (mean, 25.4 + 21.2; median, 16), but this difference did not achieve statistical significance ( P = .117). The Ki-67 index of HPV-positive AIM was significantly higher than that of normal cervix ( P < .01) and LSILs ( P < .05), and significantly lower than that of HSILs ( P < .01). The Ki-67 index of HPV-negative AIM did not differ significantly from normal cervix ( P > .05) or LSILs
(P > .05). In normal cervix, Ki-67 immunopositivity was confined to nuclei in the parabasal region (Fig 2A, B). In LSILs, the Ki-67-positive nuclei were distributed t h r o u g h o u t the lower two-thirds of the epithelium, with relative sparing of the nuclei in the u p p e r third (Fig 2C, D). The HSILs typically showed diffuse Ki-67 immunostaining of nuclei at all levels of the epithelium (Fig 2E, F). In the AIM specimens, the distribution of Ki-67positive nuclei was quite variable. Several examples of HPV-positive and HPV-negative AIM lesions are shown in Figure 1B, 1D, and 1E In some cases, the distribution o f Ki-67-positive cells resembled the pattern seen in normal cervix, whereas others displayed staining distributions similar to those seen in LSILs or HSILs. Follow-Up
NA HSIL HSIL Negative HSIL Negative HSIL
C o n c u r r e n t or subsequent cytological or tissue specimens were available from 21 of the 27 patients diagnosed with AIM. C o n c u r r e n t or subsequent HSIL was diagnosed in 13 of the 21 patients. In all cases, HSIL was diagnosed concurrently or within 3 months of the AIM diagnosis. Twelve of the 13 HSILs were identified in cervical biopsy specimens, and one was identified in a cytological specimen (Pap smear). HPV-positive AIMs were significantly more likely to have a c o n c u r r e n t or subsequent diagnosis of HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 16.7%) (P = .014 by two-tailed Fisher's exact test).
NA NA Squamous carcinoma HSIL HSIL HSIL Negative
Abbreviation: NA, not available.
349
HUMAN PATHOLOGY
Volume 30, No. 3 (March 1999)
DISCUSSION T h e wide range of Ki-67 indices and variable HPV status in AIM suggest that the histopathologic spectrum of lesions that encompass the entity we currently regard as AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (highrisk HPV-positive, low-moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). The HPV-positive AIM lesions were shown to contain the same HPV types (high- and intermediaterisk HPVs) typically identified in HSILs. Interestingly, more than half of the AIM lesions in our group contained intermediate-risk HPV types. Most HPVpositive AIMs were shown to have c o n c u r r e n t or subseq u e n t HSIL within a 3-month period. These findings support the use of HPV testing in the assessment of atypical epithelial proliferations of the cervix in which a diagnosis of AIM is considered. Given our Ki-67 immunostaining results in AIM, we would now interpret the more atypical lesions (ie, those with greater nuclear atypia and elevated Ki-67 indices) as HSIL outright, despite the paucity of mitotic figures. Notably, of 14 AIM lesions for which an HSIL diagnosis was strongly considered, 12 were HPV positive. It is also reasonable to make a diagnosis of AIM versus HSIL (or AIM versus HSIL, favor HSIL) for these more atypical AIM lesions, with the understanding that such cases should probably be managed as HSIL. Determining the appropriate m a n a g e m e n t of HPVnegative AIM lesions is significantly more problematic. We were unable to d o c u m e n t the development of HSIL in most patients with HPV-negative AIM, suggesting that a subset of AIM likely represents benign reactive conditions. All of the HPV-negative AIM lesions with low Ki-67 indices had negative follow-up. However, c o n c u r r e n t HSIL was identified in one patient with HPV-negative AIM. This particular AIM lesion showed significant elevation of the Ki-67 index. Although it is possible that HPV was not detected by PCR-based methods in this case, we think this is unlikely because in situ hybridization for detection of HPV nucleic acids was also negative (Dr Srihari Kadkhol, personal communication). Given that at least a few HPV-negative AIM lesions may be associated with HSIL, additional tissue studies and close clinical follow-up of patients with such lesions would seem prudent--particularly for those with lesions showing elevated cellular proliferation. For those pathologists without routine access to HPV typing and Ki-67 immunostaining, an alternative approach to diagnosis may be considered. We suggest a diagnosis of AIM, followed by a comment. For those AIM lesions that are highly suspicious for, but not diagnostic of, HSIL, the c o m m e n t should specify that although a benign reactive process is in the differential diagnosis, the lesion may represent an HSIL or HSIL precursor, because similar lesions, particularly when shown to be HPV positive, are strongly associated with either c o n c u r r e n t or subsequent development of more
350
overt HSILs. For those lesions that are less suspicious, the c o m m e n t should state that although HSIL is in the differential diagnosis, a benign reactive process is favored. Not surprisingly, the distinction between AIM and HSIL is often difficult and raises the possibility that the two entities may be related, at least in some cases. Although mild, moderate, and severe dysplasias of the cervix (ie, LSILs and HSILs) have been previously thought to represent a morphological and biological c o n t i n u u m in the progression of cervical cancer, more recent analyses of the natural history of cervical dysplasias suggest the possibility that LSILs and HSILs might represent distinct entities with different potential for progression to cancer (reviewed by Kiviat et apl). Therefore, not all HSILs necessarily arise from preexisting LSILs, and there may be other morphologically recognizable lesions that serve as HSIL a n t e c e d e n t s / precursors. Furthermore, in gynecologic cytology, the diagnosis of atypical squamous cells of u n d e t e r m i n e d significance composed of immature metaplastic cells is associated with a greater risk of progression to, or association with, HSILs than those atypical squamous cells of u n d e t e r m i n e d significance cases composed of mature cells. 12 We suggest the possibility that the HPVpositive AIM lesions with low to moderate elevations of the Ki-67 index might represent HSIL precursors. Clearly, larger studies o f AIM are required to confirm or refute such a hypothesis. In summary, our findings suggest that AIM, at least as recognized by pathologists at our institutions, likely represents a heterogeneous group o f lesions. Those that are positive for high- or intermediate-risk HPV types probably represent HSILs or HSIL precursors, and those that are HPV negative may represent benign reactive conditions. The strong association of concurrent or subsequent HSIL with AIM, particularly those that are HPV positive, suggests n e e d for additional tissue studies and close clinical follow-up of patients with these lesions.
A&nowledgment. The authors gratefully acknowledge the expert technical assistance of Wes Gage and George Pettis in the Ki-67 immunostaining, and Drs Robert J. Kurman and Mark Sherman for helpful discussions and critical review of the manuscript. REFERENCES 1. Crum CP, Egawa K, FuY-S, et al: Atypical immature metaplasia (AIM): A subset of human papilloma virus infection of the cervix. Cancer 51:2214-2219, 1983 2. Ward BE, Saleh AM, Williams JV, et al: Atypical immature metaplasia of the cervix: A reevaluation. Lab Invest 64:42A, 1997 (abstr) 3. Isacson C, Kessis TD, Hedrick L, et al: Both cell proliferation and apoptosis increase with lesion grade in cervical neoplasia but do not correlate with human papillomavirus type. Cancer Res 56:669674, 1996 4. Dunton CJ, van Hoeven KH, Kovatich MS, et al: Ki-67 antigen staining as an adjunct to identifying cervical intraepithelial neoplasia. Gynecol Onco164:451-455, 1997
AIM OF THE CERVIX (Geng et al) 5. A1-Saleh W, Delvenne P, Greimers R, et al: Assessment of Ki-67 antigen immunostaining in squamous intraepithelial lesions of the uterine cervix. A m J Clin Pathol 104:154-160, 1995 6. van Hoeven KH, Ramondetta L, Kovatich AJ, et al: Quantitative image analysis of MIB-1 reactivity in inflammatory, hyperplastic, and neoplastic endocervical lesions. Int J Gynecol Pathol 16:15-21, 1997 7. Kessis TD, Silberman MA, Sherman M, et al: Rapid identification of patient specimens with microsatellite DNA markers. Mod Pathol 9:183-188, 1996 8. Yoshikawa H, Kawana T, Kitagawa K, et al: Detection and typing of multiple genital human papillomaviruses by DNA amplification with consensus primers.JpnJ Cancer Res 82:524-531, 1991 9. Manos M, Ting Y, Wright A, et al: The use ofpolymerase chain reaction amplification for the detection of genital human papillomaviruses, in Furth M, Greaves M (eds): Molecular Diagnostics of
Human Cancer: Cancer Cells. Cold Spring Harbor, NY, Cold Spring Laboratory, 1989, pp 209-214 10. Payne S, Kernohan NM, Walker F: Proliferation in the normal cervix and in preinvasive cervical lesions. J Clin Pathol 49:66%671, 1996 11. Kiviat NB, Critchlow CW, Kurman RJ: Reassessment of the morphological continuum of cervical intraepithelial lesions: Does it reflect different stages in the progression to cervical carcinoma?, in Mufioz N, Bosch FX, Shah KV, Meheus A (eds): The Epidemiology of Cervical Cancer and Human Papillomavirus. Lyon, France, International Agency for Research on Cancer, 1992, pp 59-66 12. Sheils LA, Wilbur DC: Atypical squamous cells of undetermined significance: Stratification of the risk of association with, or progression to, squamous intraepithelial lesions based on morphologic subcategorization. Acta Qftol 41:1065-1072, 1997
351