Estimation of Prognoses for Cervical Intraepithelial Neoplasia 2 by p16 INK4a Immunoexpression and High-Risk HPV In Situ Hybridization Signal Types

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1 Anatomic Pathology / CIN PROGNOSES ESTIMATION Estimation of Prognoses for Cervical Intraepithelial Neoplasia by p16 INK4a Immunoexpression and High-Risk HPV In Situ Hybridization Signal Types Makiko Omori, MD, PhD, 1 Akihiko Hashi, MD, PhD, 1 Kumiko Nakazawa, PhD, Tsutomu Yuminamochi, Tetsu Yamane, MD, Shuji Hirata, MD, PhD, 1 Ryohei Katoh, MD, PhD, and Kazuhiko Hoshi, MD, PhD 1 Key Words: Cervical intraepithelial neoplasia; p16 INK4a ; Human papillomavirus; In situ hybridization; Cervical cancer DOI: 1.19/UP5PJK9RYF7BPHM Abstract The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade (CIN ) can be predicted by p16 INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 5 cases histologically diagnosed with CIN : dysplasia regressed in 8 cases; 1 cases progressed to CIN ; and CIN persisted in 11 cases. Expression of p16 INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16 INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN lesions before progression compared with those before regression. CIN cases in which moderate to strong immunoexpression of p16 INK4a and a punctate signal were observed simultaneously progressed to CIN in 1 (91%) of 11 cases. CIN cases with moderate to strong immunoexpression of p16 INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression. Uterine cervical cancer is one of the most common malignancies in women and a major cause of morbidity and mortality worldwide. Uterine cervical cancer was the eighth leading cause of cancer death in Japanese women in 4, with a mortality rate of 8.6 deaths per 1, women. 1 Interestingly, cervical cancer mortality has increased slightly since 1995, despite continued decreases since 195 in Japan. This is attributable to an increased incidence of cervical cancer in young women. The incidence of cervical dysplasia, which is potentially precancerous, has also increased in young women. Detection and treatment of cervical dysplasia in young women is important for reducing morbidity and mortality in cervical cancer, but only cases with a high risk of progression to cervical cancer should be treated, given the adverse effects of treatment on subsequent pregnancy outcomes. 4,5 Cervical intraepithelial neoplasia (CIN) has been classified into grades by the World Health Organization 6 and the General Rules for Clinical and Pathological Management of Uterine Cervical Cancer of the Japan Society of Obstetrics and Gynecology 7 : grade 1, mild dysplasia (CIN 1); grade, moderate dysplasia (CIN ); and grade, severe dysplasia and carcinoma in situ (CIN ). CIN has recently been classified into lowgrade squamous intraepithelial lesion (SIL) and high-grade SIL in the Bethesda classification system, a new set of diagnostic criteria for cytology. 8 CIN 1 is equivalent to the low-grade SIL category, and CIN and CIN are equivalent to the high-grade SIL category in the new system. The 1 Consensus Guidelines for the Management of Women With Cervical Intraepithelial Neoplasia by the American Society for Colposcopy and Cervical Pathology recommend that patients with CIN 1 be followed up without treatment if colposcopic examination yields satisfactory results because most cases of CIN 1 regress spontaneously without treatment The guidelines recommend that patients with 8 Am J Clin Pathol 7;18:8-17 Downloaded 8 from DOI: 1.19/UP5PJK9RYF7BPHM on 14 February 18

2 Anatomic Pathology / ORIGINAL ARTICLE CIN and CIN receive treatment using excisional or ablative modalities because these methods reduce the incidence and mortality caused by invasive cervical cancer. 9 However, whether treatment is necessary for CIN has been controversial in Japan. 1 Most cases of CIN have been treated after progression to CIN in Japan because regression of CIN is considered relatively common A long-term follow-up study by Nasiell et al 1 observed that 54% of CIN cases regressed, 16% persisted, and % progressed. Other investigators have observed regression rates from CIN to normal of 5% to 57% and progression rates from CIN to CIN or worse of 1% to % Even if the progression rate of CIN is higher than the regression rate, treating all cases of CIN might represent overtreatment. High-risk human papilloma virus (HPV) infection is known to be the most important event in the malignant transformation of cervical epithelium. 19, E6 and E7 oncoproteins from HPV inhibit the tumor suppressor functions of p5 and retinoblastoma protein, respectively. 1 The E7 oncoprotein has been shown to bind to retinoblastoma protein, resulting in the release of EF transcription factors that regulate cell proliferation. EF overexpression leads to an inhibition of cyclin D1 dependent kinase activity and, consequently, induces expression of a p16 INK4a -related transcript., In other words, increased expression of p16 INK4a indicates an HPV-induced abnormality of the cell cycle and immortalization of HPVinfected cells. This is observed in neoplastic cervical epithelial cells. As a result, p16 INK4a overexpression is considered useful as a biomarker of malignant transformation by HPV. 4,5 Integration of HPV DNA into host-cell DNA occurs early in cancer development and represents an important event in malignant transformation of cervical epithelium. 6 The integrated genome is detected as a punctate signal in the nuclei of neoplastic cells by using in situ hybridization (ISH), whereas the episomal genome is detected as a diffuse signal throughout the nuclei. 7,8 The punctate signal of HPV is found in highgrade dysplastic lesions and invasive cancers. 9, A highly sensitive ISH method with signal amplification by tyramide has been developed and allows detection of low-copy DNA. 8,1 The punctate signal detected by HPV ISH is also considered a useful biomarker of malignant transformation by HPV. 8,, We examined whether progression of CIN could be predicted by p16 INK4a overexpression and a high-risk HPV ISH signal. If high-risk cases of progression could be detected among CIN cases, this would enable appropriate treatment only for cases that would actually benefit from treatment. Japan. Diagnoses of CIN were reviewed independently by pathologists (A.H., T.Y., and R.K.). Only cases in which complete diagnostic accord was reached among the pathologists were selected for this study. The following cases were excluded from the study: pregnant patients; patients undergoing immunosuppressant therapy; and patients diagnosed with progression in the first months after initial diagnosis because the worst part of a lesion might have been missed in the first colposcopy. In addition, cases of CIN treated by conization and 1 cases of invasive cervical cancer diagnosed histologically as squamous cell carcinoma (SCC) were randomly selected from the pathology records of our institution. Cases of CIN were divided into groups: 1 (n = 8), in which atypical cells disappeared during later follow-up; (n = 11), in which CIN continued for more than years; and (n = 1), in which surgery was performed owing to progression to CIN during later follow-up. Cases of CIN 1 and CIN were followed up by cervical smears and colposcopy every to 4 months, and biopsies were performed when progression was suspected based on cytologic or colposcopic examination. When a dysplastic cell was not found in at least consecutive examinations, an assessment of regression was made. Informed consent was obtained from all patients. Immunohistochemical Analysis, ISH, and Polymerase Chain Reaction Consecutive sections were prepared from formalin-fixed, paraffin-embedded tissue samples obtained from biopsies and resected specimens from surgery. One specimen was used for standard histologic analysis, and the other specimens were used for further analyses. One of these was immunostained using a CINtec p16 INK4a Histology kit (DAKO Japan, Tokyo) to detect Materials and Methods Case Selection Cases comprised 5 cases of CIN 1 and 5 cases of CIN Image 1 diagnosed from colposcopically directed biopsies at the Faculty of Medicine, University of Yamanashi, Yamanashi, Image 1 A case of cervical intraepithelial neoplasia diagnosed from colposcopically directed biopsy (H&E, 5). Downloaded from on 14 February 18 Am J Clin Pathol 7;18: DOI: 1.19/UP5PJK9RYF7BPHM 9

3 Omori et al / CIN PROGNOSES ESTIMATION expression of p16ink4a according to the directions of the manufacturer. ISH was performed on each specimen using a GenPoint Catalyzed Signal Amplification System (DAKO Japan) for highrisk HPV (types 16, 18, 1,, 5, 9, 45, 51, 5, 56, 58, 59, and 68) according to the instructions of the manufacturer. GenPoint has been evaluated as the most sensitive method for detecting 1 or copies of HPV DNA on isolated cells by ISH.1 The HPV subtype was examined at initial diagnosis and after progression to CIN in CIN group. The HPV subtype was determined by using the HPV Typing Set (Takara Bio, Shiga, Japan), a primer set for polymerase chain reaction (PCR) specifically designed to identify HPV types 6, 11, 16, 18, 1,, 5, 5b, and 58 in genomic DNA. Evaluation of Immunohistochemical and ISH Results The degree of p16ink4a immunoreactivity was graded as follows: (negative), fewer than 1% of cells positive; 1+ (weak), weak in the nuclei and faint in cytoplasm Image A ; A B Image Immunohistochemical staining for p16ink4a. The degree of p16ink4a immunoreactivity was graded as follows: negative; 1+, weak in nuclei and faint in cytoplasm (A, 5); +, moderate in nuclei and weak to moderate in cytoplasm (B, 5); and +, strong in nuclei and cytoplasm (C, 5). C 1 + (moderate), moderate in the nuclei and weak to moderate in cytoplasm Image B ; and + (strong), strong in the nuclei and cytoplasm Image C. Immunostaining distribution patterns were classified according to the evaluation methods described by Klaes et al5 as sporadic (isolated positive cells, but <5%) Image A, focal (small cell clusters, but <5% positive cells) Image B, or diffuse (>5% positive cells) Image C and thickness into one third of Image 4A, two thirds of Image 4B, and throughout Image 4C the epithelium. HPV ISH signal types can be classified into diffuse and punctate signals, representing episomal and integrated HPV DNA, respectively. Cases in this study were classified into diffuse Image 5A, punctate Image 5C, and mixed Image 5B signals that combined both, and into (<1%), 1+ (<1%), + (1%-5%), and + (>5%) according to the count of signalpositive cells. Areas of greatest p16ink4a immunostaining were evaluated. In the same areas, HPV ISH signal types and counts of signal-positive cells were evaluated. Am J Clin Pathol 7;18:8-17 Downloaded 1 from DOI: 1.19/UP5PJK9RYF7BPHM on 14 February 18

4 Anatomic Pathology / ORIGINAL ARTICLE Identifications of p16ink4a immunoreactivity, HPV ISH signal types, and the count of signal-positive cells were reviewed independently by at least pathologists. In cases of discordant results, a third pathologist blinded to the results of previous diagnostic findings was consulted. When of pathologists gave the same judgment, that judgment was adopted. Statistics The κ statistic was used to assess degree of interobserver agreement for interpretation of p16ink4a immunostaining and HPV ISH signal. Associations between variables were assessed using nonparametric tests such as the Spearman correlation coefficient by rank test, Kruskal-Wallis test, χ test for independence, and the Mann-Whitney U test. P values of less than.5 were considered statistically significant. A C Results The mean age of patients was 6.6 years for CIN 1 (range, -61 years), 6.1 years for CIN group 1 (range, 1964 years), 4.5 years for CIN group (range, -47 years), 9. years for CIN group (range, 8-51 years), 9.4 years for CIN (range, -6 years), and 4.1 years for SCC (range, 6-64 years). The median duration of follow-up was 41.5 months for CIN 1 (range, 7-81 months), 45.5 months for CIN group 1 (range, months), 47. months for CIN group (range, 4-79 months), and 7 months for CIN group (range, 18-5 months). In all 5 cases of CIN 1, dysplastic lesions disappeared at a median of 1.5 months after initial diagnosis (range, -57 months). Dysplastic lesions disappeared within years in 18 (7%) of the CIN 1 cases. In CIN group 1, lesions disappeared at a median of 7.5 months after initial diagnosis (range, -9 months) and within years in 4 (86%) of 8 cases. In B Image Immunohistochemical staining for p16ink4a. Distribution patterns of p16ink4a immunoreactivity were graded as follows: negative; sporadic, scattered staining (A, 5); focal, focal staining (B, 5); and diffuse, diffuse staining (C, 5). Am J Clin Pathol 7;18:8-17 Downloaded from on 14 February DOI: 1.19/UP5PJK9RYF7BPHM 11 11

5 Omori et al / CIN PROGNOSES ESTIMATION A B Image 4 Immunohistochemical staining for p16ink4a. The thickness of p16ink4a immunoreactivity in the cervical epithelium was graded as follows: negative; lower one third (A, 5); lower two thirds (B, 5); and full thickness (C, 5). C CIN group, progression to CIN occurred at a median of 18 months after initial diagnosis (range, 6-9 months) and within years in 11 (85%) of 1 cases. In 1 case of CIN, progression to CIN occurred after a long duration, 9 months, after initial diagnosis. The degree, distribution pattern, and thickness of p16ink4a immunoexpression were compared with grade of dysplasia Table 1. Immunoexpression of p16ink4a of 1+ (weak) or more was observed in 6% of CIN 1 (15/5), 87% of CIN (45/5), and 1% of CIN (/) and SCC (1/1) cases. Moderate to strong immunoexpression of p16ink4a was observed in 8% of CIN 1 (/5), 7% of CIN (14/5), 9% of CIN (18/), and 1% of SCC (1/1) cases. The degree of p16ink4a immunoreactivity increased significantly with worsening grade of CIN (P <.1). Diffuse distribution of p16ink4a immunoexpression was significantly associated with CIN grade (P <.1) and was observed in 6% of CIN 1 (9/5), 7% of CIN (8/5), and 1% of 1 Am J Clin Pathol 7;18:8-17 Downloaded 1 from DOI: 1.19/UP5PJK9RYF7BPHM on 14 February 18 CIN (/) and SCC (1/1) cases. Thickness of p16ink4a immunoreactivity in the epithelium significantly increased with worsening CIN grade (P <.1). Immunoexpression of p16ink4a was observed in the lower third of the epithelium in 5% of CIN 1 (8/15), in the lower two thirds in 56% of CIN (5/45), and throughout the epithelium in 1% of CIN (/) and SCC (1/1) cases. Interobserver agreement rates in grading p16ink4a immunoreactivity for CIN1 and CIN were excellent (degree, κ =.9; distribution pattern, κ =.87; and thickness, κ =.9), whereas rates for the diagnostic interpretation of H&E staining for CIN 1 and CIN were moderate (CIN 1, pairwise κ,.6.71; CIN, pairwise κ,.4-.65). Diagnoses of CIN and SCC displayed complete accord among the pathologists. The prognosis for CIN was significantly dependent on degree of p16ink4a immunoreactivity (P <.1). The degree of p16ink4a immunoexpression at initial diagnosis was significantly stronger in CIN group than in CIN groups 1 or

6 Anatomic Pathology / ORIGINAL ARTICLE A B Image 5 A high-risk human papillomavirus in situ hybridization signal was observed in the nuclei of dysplastic cells. Signal patterns were classified as diffuse (A, 1), mixed (B, 1), or punctate (C, 1) signal. A diffuse signal was confined to the upper layer of the epithelium, whereas a punctate signal was predominantly observed in the lower layer of the epithelium. C Table 1 Relation Between p16ink4a Overexpression and CIN Grade and Prognosis of CIN Degree* CIN 1 (n = 5) CIN (n = 5) CIN subgroup 1 (n = 8) (n = 11) (n = 1) CIN (n = ) SCC (n = 1) Distribution Pattern Thickness Negative Sporadic Focal Diffuse Lower Third Two Thirds Full CIN, cervical intraepithelial neoplasia; SCC, squamous cell carcinoma. * The degree of p16ink4a immunoreactivity was graded as follows: negative, <1% of cells positive; 1+, weak in nuclei and faint in cytoplasm; +, moderate in nuclei and weak to moderate in cytoplasm; and +, strong in nuclei and cytoplasm. CIN 1 vs CIN vs CIN, P <.1; CIN subgroup 1 vs vs, P <.1. Immunostaining distribution patterns were classified as follows: sporadic, isolated positive cells, but <5%; focal, small cell clusters, but <5% positive cells; or diffuse, >5% positive cells. CIN 1 vs CIN vs CIN, P <.1; CIN subgroup 1 vs vs, not significant. Thickness was classified as one third or two thirds of, and throughout the epithelium (full). CIN 1 vs CIN vs CIN, P <.1; CIN subgroup 1 vs vs, not significant. Am J Clin Pathol 7;18:8-17 Downloaded from on 14 February 18 1 DOI: 1.19/UP5PJK9RYF7BPHM 1 1

7 Omori et al / CIN PROGNOSES ESTIMATION (P <.5). No significant differences in degree of p16 INK4a immunoreactivity were identified in lesions at initial diagnosis between CIN group and CIN or between CIN group 1 or group and CIN 1. Cases of CIN showing equal p16 INK4a immunoreactivity with CIN progressed to CIN. No lesions showing strong (+) immunoexpression of p16 INK4a were detected in CIN 1. All 6 cases of CIN showing strong immunoexpression progressed to CIN. No cases of CIN progressed to CIN when p16 INK4a immunoexpression was observed in sporadic or focal distribution or in the lower third of the epithelium at initial diagnosis. High-risk HPV ISH signals were detected in 8% of CIN 1 (7/5), 6% of CIN (1/5), and 1% of CIN (/) and SCC (1/1) cases Table. Punctate signals were observed in none of 5 CIN 1, 1% of CIN (11/5), 6% of CIN (1/), and 9% of SCC (9/1) cases. Punctate signals and HPV signal-positive cells increased with worsening grade of CIN (P <.1 each). Interobserver agreement rates were excellent for assessment of HPV ISH signal (count of HPV ISH signal-positive cells, κ =.96; signal type, κ =.95). Punctate signals and signal-positive cells were significantly increased in CIN group compared with group 1 or group on initial diagnosis of CIN (punctate signal, P <.1; signal-positive cell, P <.5). No differences in signal type or count of signal-positive cells were noted between CIN group and CIN at initial diagnosis. CIN in which HPV signalpositive cells were increased and punctate signal was detected to the same extent as in CIN progressed to CIN. In 11 cases of CIN in which only a punctate signal was detected, CIN progressed to CIN in 8 cases (7%), persisted in cases (7%), and regressed in no cases (%). Dysplastic lesions regressed in all 6 cases of CIN in which only a diffuse signal was detected. In 14 cases of mixed signal, dysplastic lesions progressed in cases (1%) and regressed in 9 cases (64%). When individual CIN group cases were compared, no differences were seen between before and after progression to CIN with regard to p16 INK4a immunoreactivity, count of HPV signal-positive cells, or signal type Table. The same HPV subtypes (16, 1, 5, and 58) were detected between initial diagnosis and after progression to CIN in 4 cases. HPV subtypes changed between initial diagnosis and after progression in 4 cases; in 1 case, from HPV- to multiple types (1,, and 5); in 1 case, from HPV-5 to HPV-1 and HPV-5; in 1 case, from multiple HPV types (1,, and 5) to HPV- 1; and in 1 case, from multiple HPV types (1, 5, and 58) to HPV-58. We found no significant differences with regard to HPV subtype before and after progression to CIN (Table ). The degree of p16 INK4a immunoexpression correlated significantly with ISH signal types in CIN cases (P <.1) Table 4. Prognoses for CIN were studied in relation to p16 INK4a immunoexpression and high-risk HPV ISH signal. When strong immunoexpression of p16 INK4a or only a punctate signal was detected, no cases of CIN regressed (progression, 9 of 1; persistence, of 1). When both moderate to strong immunoexpression of p16 INK4a and a punctate signal were detected, 91% of CIN cases (1/11) progressed to CIN (P <.1) Table 5. Discussion Recent molecular biologic and epidemiologic studies have demonstrated that infection with high-risk HPV represents the principal cause of cervical cancer and CIN. 4,5 In this study, high-risk HPV signals using ISH were detected in 8% of CIN 1, 6% of CIN, 1% of CIN, and 1% of SCC cases (Table ). Risk of CIN progression is also reportedly dependent on the type of HPV. 17,6 However, a high percentage of healthy women infected with high-risk HPV do not develop cervical cancer. A 1-year cohort study of women with a positive high-risk HPV test showed the frequency of CIN or cancer was 6.9% for the entire follow-up period. 7 Both host and viral genetic factors are recognized as having a role in carcinogenesis. 8 Table Relation Between High-Risk HPV In Situ Hybridization Signal and CIN Grade and Prognosis of CIN HPV Signal Positive Cell * HPV Signal Pattern Negative Diffuse Mixed Punctate CIN 1 (n = 5) CIN (n = 5) CIN subgroup 1 (n = 8) (n = 11) (n = 1) CIN (n = ) SCC (n = 1) CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; SCC, squamous cell carcinoma. * Categories were as follows: negative, <1%; 1+, <1%; +, 1%-5%; and +, >5% of cells. CIN 1 vs CIN vs CIN, P <.1; CIN subgroup 1 vs vs, P <.5. Signal types were classified as follows: diffuse, representing episomal HPV DNA; punctate, representing integrated HPV DNA; mixed, a combination of diffuse and punctate. CIN 1 vs CIN vs CIN, P <.1; CIN subgroup 1 vs vs, P < Am J Clin Pathol 7;18:8-17 Downloaded 14 from DOI: 1.19/UP5PJK9RYF7BPHM on 14 February 18

8 Anatomic Pathology / ORIGINAL ARTICLE Table Comparison of p16 INK4a Overexpression, High-Risk HPV ISH Signal, and HPV Type Before and After Progression of CIN to CIN * p16 INK4a High-Risk HPV ISH Degree Distribution Thickness Signal Type Signal-Positive Cell HPV Type Age (y) CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN D D / Full P P D D / Full P P D D / Full M 1+ 1,, D D / Full P P D D Full Full P P D D / Full P D D Full Full P P D D Full Full M M D D Full Full M M D D Full Full P M ,, D Full D D Full Full P P , D D / Full P P , 5, CIN, cervical intraepithelial neoplasia; D, diffuse pattern; HPV, human papillomavirus; ISH, in situ hybridization; M, mixed signal; P, punctate signal; SCC, squamous cell carcinoma. * The degree of p16 INK4a immunoreactivity was graded as follows:, <1% of cells positive; 1+, weak in nuclei and faint in cytoplasm; +, moderate in nuclei and weak to moderate in cytoplasm; and +, strong in nuclei and cytoplasm. Diffuse immunostaining indicates >5% positive cells. Thickness was classified as one third or two thirds of or throughout the epithelium (full). Signal types were classified as punctate, representing integrated HPV DNA, or mixed, a combination of diffuse (representing episomal HPV DNA) and punctate. Categories for signal-positive cells were as follows:, <1%; 1+, <1%; +, 1%-5%; and +, >5% of cells. At initial diagnosis. Table 4 Relation of the Degree of p16 INK4a Immunoexpression and High-Risk HPV ISH Signal Type in CIN Cases * ISH Negative Diffuse Mixed Punctate Total/Percentage p16 INK4a Negative 7 (1) () () () 7 (1)/ (1) () 1 () 4 (1) 1 ()/7 + () () () () 8 (4)/5 + () () 1 (1) 5 (5) 6 (6)/1 Total/percentage 1 ()/1 6 ()/ 14 ()/1 11 (8)/7 5 (1)/5 CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; ISH, in situ hybridization. * Data are given as number of CIN cases (number that progressed to CIN ) unless otherwise indicated. The degree of p16 INK4a immunoreactivity was graded as follows: negative, <1% of cells positive; 1+, weak in nuclei and faint in cytoplasm; +, moderate in nuclei and weak to moderate in cytoplasm; and +, strong in nuclei and cytoplasm. Signal types were classified as follows: diffuse, representing episomal HPV DNA; punctate, representing integrated HPV DNA; or mixed, a combination of diffuse and punctate. P <.1 for the degree of p16 INK4a immunoreactivity vs high-risk HPV ISH signal type in CIN cases. Percentage of cases in the total column that progressed to CIN. Percentage of cases in the total row that progressed to CIN. Table 5 Relation Between Prognosis of CIN and High-Risk HPV In Situ Hybridization Signal Type and p16 INK4a Immunoreactivity in 5 Cases of CIN * Punctate Signal Negative Positive (Punctate and Mixed) p16 INK4a (-1+) (n = 4) p16 INK4a (+-+) (n = ) p16 INK4a (-1+) (n = 14) p16 INK4a (+-+) (n = 11) Regression 16 (67) (1) 8 (57) 1 (9) Persistence 6 (5) () 5 (6) () Progression (8) () 1 (7) 1 (91) CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus. * Data are given as number (percentage). The degree of p16 INK4a immunoreactivity was graded as follows:, <1% of cells positive; 1+, weak in nuclei and faint in cytoplasm; +, moderate in nuclei and weak to moderate in cytoplasm; and +, strong in nuclei and cytoplasm. Punctate represents integrated HPV DNA, and mixed, a combination of diffuse (representing episomal HPV DNA) and punctate. P <.1 for prognosis vs high-risk HPV ISH signal type and the degree of p16 INK4a immunoreactivity in CIN cases. Downloaded from on 14 February 18 Am J Clin Pathol 7;18: DOI: 1.19/UP5PJK9RYF7BPHM 15

9 Omori et al / CIN PROGNOSES ESTIMATION The development of analyses for p16 INK4a overexpression and integration of HPV DNA has enabled determination of the physical status of HPV DNA and cervical carcinogenesis. 4,5,8, In the present study, moderate to strong immunoexpression of p16 INK4a was observed in 8% of CIN 1, 7% of CIN, 9% of CIN, and 1% of SCC cases (Table 1). A high-risk HPV punctate signal, which indicates HPV integration into the genome, was detected in % of CIN 1, 1% of CIN, 6% of CIN, and 9% of SCC cases by using ISH methods (Table ). Expression of p16 INK4a and a high-risk HPV punctate signal correlated significantly with increased CIN grade. Kalof et al 9 reported similar results and the usefulness of either or both tests in confirming CIN or. Previous studies have demonstrated a high level of interobserver variability in the histologic diagnosis of CIN, particularly CIN. 4,41 Klaes et al 4 reported that p16 INK4a immunostaining in cervical biopsy specimens can reduce interobserver disagreement in the interpretation of cervical lesions. The present study also showed that interobserver agreement for p16 INK4a immunostaining was better than that for H&E staining in diagnoses of CIN 1 and CIN. As a result, p16 INK4a seems to be a very useful marker allowing precise identification of cervical lesions. The detection of p16 INK4a overexpression and a high-risk HPV punctate signal has been suggested to be useful for predicting progression of CIN. 4 However, few studies have investigated progression of CIN using p16 INK4a immunoreactivity and high-risk HPV ISH signals. The present study revealed that p16 INK4a overexpression and high-risk HPV punctate signals correlate not only with CIN grade but also with prognosis for CIN. Stronger immunoexpression of p16 INK4a and higher frequency of expression for high-risk HPV punctate signals were seen in CIN lesions before progression compared with those before regression (Tables 1 and ). All cases of CIN showing strong immunoexpression of p16 INK4a at initial diagnosis progressed to CIN (Table 4). In 91% of CIN cases showing moderate to strong immunoexpression of p16 INK4a and a punctate signal (punctate only or mixed signal), progression to CIN occurred (Table 5). CIN lesions showing expression of p16 INK4a and a punctate signal equal to that of CIN progressed to CIN during later follow-up. Conversely, CIN lesions showing expression of these biomarkers equal to that of CIN 1 spontaneously regressed to normal. Our results suggest that lesions histologically diagnosed as CIN show various degrees of malignant transformation from CIN 1 to CIN. Moreover, few studies have investigated progression of CIN with an ISH mixed signal a combined punctate and diffuse signal. Whether detection of a mixed signal represents a process of transition from an episomal HPV genome to an integrated genome or the inverse or some other phenomenon has not been reported. In our cases with mixed signals, all cases with weak p16 INK4a immunoexpression regressed to normal. Consequently, simultaneous identification of p16 INK4a overexpression and a high-risk HPV punctate signal at initial diagnosis may enable prediction of progression of CIN. Even if a woman is infected with high-risk HPV, she will have a smaller risk of developing cervical cancer if weak overexpression of p16 INK4a or a diffuse ISH signal is detected in the CIN lesion, although these findings need to be confirmed by further larger prospective studies with longitudinal follow-up. Cases of CIN showing moderate to strong immunoexpression of p16 INK4a and a high-risk HPV punctate signal at initial diagnosis seem to warrant immediate treatment given the high risk of progression. From the 1 Department of Obstetrics and Gynecology, Clinical Laboratory, and Department of Human Pathology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan. Address reprint requests to Dr Omori: Dept of Obstetrics and Gynecology, Faculty of Medicine, University of Yamanashi, 111 Shimokato, Chuo, Yamanashi, , Japan. Acknowledgments: We gratefully acknowledge the contributions of Takeshi Endo and Makoto Osada, Clinical Laboratory, Faculty of Medicine, University of Yamanashi, for performing PCR; Naoko Sakamoto, Clinical Laboratory, Faculty of Medicine, University of Yamanashi, for help with the performance of ISH; and Shin-ichi Murata, MD, PhD, Department of Human Pathology, Faculty of Medicine, University of Yamanashi, for histopathologic analyses. References 1. Ministry of Health, Labour and Welfare (MHLW) statistical database, Japanese. Available at toukei/data/1/4/toukeihyou/498/t19/mc5 _6.html. Accessed August 17, 6.. Ikeda K, Kamimura H. Studies on characteristics of cause of death in Japan. Available at SAGE/SAGE98/shibouto.pdf. Accessed August 17, 6.. Ito T, Ishizuka T, Suzuki K, et al. Cervical cancer in young Japanese women. Arch Gynecol Obstet. ;64: Sadler L, Saftlas A, Wang W, et al. Treatment for cervical intraepithelial neoplasia and risk of preterm delivery. JAMA. 4;91: Samson SL, Bentley JR, Fahey TJ, et al. The effect of loop electrosurgical excision procedure on future pregnancy outcome. Obstet Gynecol. 5;15: Tavassoli FA, Devilee P, eds. World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Lyon, France: IARC Press; : Japan Society of Obstetrics and Gynecology, The Japanese Society of Pathology, and Japan Radiological Society. The General Rules for Clinical and Pathological Management of Uterine Cervical Cancer, October nd ed. 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