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About 13,000 women will get invasive cervical cancer in the year 2000. Despite the success of the Pap smear, cervical carcinoma is still a very deadly although treatable and curable disease if caught early enough. Yet, about 5,000 women will still die from this disease every year. The human papilloma virus (HPV) has been definitely linked as the main etiologic agent for the disease. Cervical cancer follows a progression of dysplasia to in situ carcinoma to invasive carcinoma.

CIN 1-2 22 7 years to CIS 50%
CIN 3 28 7-10 years to microinvasion 66%
Microinvasion 41 2 years to invasive carcinoma 100%


About 70 virotypes have been identified for HPV. About 23 affect the cervix and genital tract and currently the types documented as conferring a higher risk of cervical cancer include: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Types 6 and 11 are associated with condyloma and are not thought to be oncogenic. Type 16 is found in about half of all cervical cancer cases in the US and Europe.

This article deals with squamous cell carcinoma of the cervix, the most common histologic subtype. For information regarding cervical adenocarcinoma, please visit this link.


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Incidence of cervical squamous intraepithelial lesions associated with HIV serostatus, CD4 cell counts, and human papillomavirus test results.

Harris TG, Burk RD, Palefsky JM, Massad LS, Bang JY, Anastos K, Minkoff H, Hall CB, Bacon MC, Levine AM, Watts DH, Silverberg MJ, Xue X, Melnick SL, Strickler HD.

Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
JAMA. 2005 Mar 23;293(12):1471-6. Abstract quote  

CONTEXT: Recent cervical cancer screening guidelines state that the interval between screenings can be safely extended to 3 years in healthy women 30 years or older who have normal cytology results and have negative test results for oncogenic human papillomavirus (HPV) DNA.

OBJECTIVE: To determine the incidence of squamous intraepithelial lesions (SILs) in HIV-seropositive women with normal cytology results, by baseline HPV DNA results.

DESIGN, SETTING, AND PATIENTS: Participants were HIV-seropositive (n = 855; mean age, 36 years) and HIV-seronegative (n = 343; mean age, 34 years) US women with normal baseline cervical cytology who were enrolled in the Women's Interagency HIV Study (WIHS), a large, multi-institutional prospective cohort study. Since their recruitment during 1994-1995, WIHS participants have been followed up semi-annually with repeated Pap smears for a median of 7 years.

MAIN OUTCOME MEASURE: The cumulative incidence of any SIL and high-grade SIL or cancer (HSIL+) was estimated according to baseline HPV DNA results, stratified by HIV serostatus and CD4 T-cell count. RESULTS: Development of any SIL in women with negative HPV results (both oncogenic and nononcogenic) at 2 years was as follows: in HIV-seropositive women with CD4 counts less than 200/microL, 9% (95% CI, 1%-18%); with CD4 counts between 200/muL and 500/microL, 9% (95% CI, 4%-13%); and with CD4 counts greater than 500/microL, 4% (95% CI, 1%-7%). The CIs for these estimates overlapped with those for HIV-seronegative women with normal baseline cytology who were HPV-negative (3%; 95% CI, 1%-5%), indicating that at 2 years, there were no large absolute differences in the cumulative incidence of any SIL between groups. Furthermore, no HPV-negative participants in any group developed HSIL+ lesions within 3 years. Multivariate Cox models showed that on a relative scale, the incidence of any SIL among HIV-seropositive women with CD4 counts greater than 500/microL (hazard ratio [HR], 1.2; 95% CI, 0.5-3.0), but not those with CD4 counts less than or equal to 500/microL (HR, 2.9; 95% CI, 1.2-7.1), was similar to that in HIV-seronegative women.

CONCLUSION: The similar low cumulative incidence of any SIL among HIV-seronegative and HIV-seropositive women with CD4 counts greater than 500/microL and who had normal cervical cytology and HPV-negative test results suggests that similar cervical cancer screening practices may be applicable to both groups, although this strategy warrants evaluation in an appropriate clinical trial.
HPV N Engl J Med 1999;341:1633-1638
Lancet 2000;355:2189-2193
Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.

1Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.


Mod Pathol. 2006 Dec 22; [Epub ahead of print] Abstract quote

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression.

To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89-9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories.

Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.
Distribution of HPV genotypes in 282 women with cervical lesions: evidence for three categories of intraepithelial lesions based on morphology and HPV type.

1Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.


Mod Pathol. 2006 Dec 22; [Epub ahead of print] Abstract quote

Previously we found differences in the distribution of the individual human papillomavirus types in cervical cancers and high-grade squamous intraepithelial lesions. This suggested that there were differences in risk for progression of high-grade squamous intraepithelial lesions that were related to human papillomavirus type within the category of oncogenic genotypes.

In this work, we add additional cases including low-grade squamous intraepithelial lesions. ThinPrep((R)) samples from 282 squamous intraepithelial lesions and invasive cervical cancers were categorized morphologically by consensus interpretation and genotyped for 27 individual human papillomavirus types by polymerase chain reaction-based reverse line blot analysis using PGMY09/PGMY11 consensus primers for the L1 open reading frame.

The 27 human papillomavirus types were divided into three categories: high risk 16, 18, 31, 45; intermediate risk 33, 35, 39, 51, 52, 56, 58, 59, 68, 73, 82, 83; and low risk: 6, 11, 26, 40, 42, 53, 54, 55, 57, 66, and 84. Of the 282 cases of cancer and squamous intraepithelial lesions, 95.7% were positive for one or more of 27 human papillomavirus types and 38.7% had two or more genotypes.

Three major categories of squamous intraepithelial lesions were identified based upon the combination of consensus diagnosis and human papillomavirus category: (1) high-grade squamous intraepithelial lesions associated with high-risk human papillomavirus types that appear to be at increased risk for progression to carcinoma; (2) squamous intraepithelial lesions (typically low-grade intraepithelial lesions and high-grade lesions consistent with moderate dysplasia) associated with intermediate risk human papillomavirus types with limited or indeterminate risk for progression; (3) low-grade squamous intraepithelial lesions associated with low-risk human papillomavirus types with little or no risk for progression. Only a subset of human papillomavirus genotypes commonly considered to be oncogenic were closely associated with invasive cervical cancer and high-grade squamous intraepithelial lesions classed as severe dysplasia. Other oncogenic types were closely associated with high-grade squamous intraepithelial lesions of moderate dysplasia and low-grade squamous intraepithelial lesions.

This suggests that risk for progression to invasion in squamous intraepithelial lesions is closely related to human papillomavirus genotype. Knowledge of the associated human papillomavirus type in women with morphologic squamous intraepithelial lesions may help to clarify risk for progression.
Comparison of human papillomavirus genotypes in high-grade squamous intraepithelial lesions and invasive cervical carcinoma: evidence for differences in biologic potential of precursor lesions.

Zuna RE, Allen RA, Moore WE, Mattu R, Dunn ST.

1Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Mod Pathol. 2004 Nov;17(11):1314-22. Abstract quote  

High-grade squamous intraepithelial lesions of the cervix are heterogeneous in their invasive potential. Comparison of human papillomavirus types between invasive cervical carcinoma and high-grade squamous dysplasia may provide insight into this biological variability.

Liquid-based Pap specimens from 55 high-grade intraepithelial lesions and 47 invasive cervical carcinomas were analyzed by reverse line blot for 27 human papillomavirus types designated high, intermediate, or low risk. Human papillomavirus DNA was present in all high-grade intraepithelial lesions (23 different types) and in 94% (13 types) of invasive carcinomas. High-risk types were present in 81% of invasive carcinomas compared to 58% of high-grade intraepithelial lesions. Severe dysplasias harbored more (79%) high-risk human papillomaviruses as compared to moderate dysplasias (37%). In 40% of high-grade dysplasia cases (59% of moderate dysplasias; 21% of severe) and 13% of invasive carcinomas, intermediate-risk genotypes were identified in the absence of high-risk HPV types. Human papillomavirus 16 was the most common type in all categories, including 47% of high-grade squamous dysplasias (26% moderate; 68% severe) and 61% of invasive carcinomas. Both high-risk type (P=0.0004) and type 16 (P=0.0007) human papillomaviruses were positively associated with increasing severity of diagnosis.

The heterogeneous nature of high-grade squamous dysplasias as compared to invasive carcinoma is evident by the wider spectrum of associated human papillomavirus types. Likewise, moderate dysplasia appears to be more heterogeneous in viral type than severe dysplasia.

Moderate cases were more often associated with intermediate-risk types, while high-risk types were more prevalent in severe dysplasias and invasive cancers. Moderate dysplasia cases harboring viral types infrequently found in cancers may have a low risk for progression. Human papillomavirus genotyping of high-grade squamous intraepithelial lesions may be important in assessing risk for progression to invasion.
The site of infection and ethnicity of the patient influence the biological pathways to HPV-induced mucosal cancer.

Li W, Thompson CH, Cossart YE, O'Brien CJ, Liu J, Scolyer RA, Carter JR, Dalrymple C, Rose BR.

Sydney Head and Neck Cancer Institute, Royal Prince Alfred Hospital, Sydney, Australia.
Mod Pathol. 2004 Sep;17(9):1031-7. Abstract quote  

High-risk human papillomaviruses are the causative agents of cervical cancer and are also believed to be aetiologically involved in a subset of squamous cell carcinomas of the head and neck region, especially the tonsil. Cervical cancers arise through disruption of the pathways of p53 and the product of the retinoblastoma gene by the human papillomavirus oncoproteins E6 and E7. It is generally assumed that the same pathways are involved in human papillomavirus-induced carcinogenesis at other mucosal surfaces. However, the patterns of expression of cell cycle proteins targeted by human papillomavirus E6 and E7 in cancers from different anatomic sites have been inconsistent, due to either biologic or technological factors.

In this study, 73 human papillomavirus, 16-positive cervical squamous cell carcinomas (35 from Australian and 38 from Chinese women) were analysed for the expression of p53, pRb, p16(INK4A), p21(CIP1/WAF1), p27(KIP1) and cyclin D1 by semiquantitative immunohistochemistry. Cervical cancers from Chinese women were found to be significantly more likely to overexpress p53, pRb, p21 and p27 than their Australian counterparts. These findings were compared with those from 31 human papillomavirus 16-positive tonsillar squamous cell carcinomas, all of Australian origin, tested using the same methodology. Comparisons of the tonsillar and combined cervical data showed that tonsillar cancers were significantly more likely to be p53-positive, whereas cervical cancers were significantly more likely to overexpress pRb, p16 and p27.

When the tonsillar data were compared with cervical data from Australian women, the associations for p53 and pRb remained. These findings represent new evidence that the molecular pathways to human papillomavirus-induced mucosal cancer may be influenced by anatomic location and ethnicity.

Human papillomavirus typing of rare cervical carcinomas.

Matthews-Greer J, Dominguez-Malagon H, Herrera GA, Unger J, Chanona-Vilchis J, Caldito G, Turbat-Herrera EA.

Department of Pathology, Louisiana State University Health Sciences, Shreveport 71130-3932, USA.
Arch Pathol Lab Med. 2004 May;128(5):553-6. Abstract quote  

CONTEXT: Most cervical tumors are classified as squamous cell carcinoma or adenocarcinoma, both of which are associated with persistent human papillomavirus (HPV) infection. Although other (rare) types represent less than 5% of all cervical carcinomas, it is necessary that these more unusual tumors be studied in the current era of papillomavirus vaccine development, especially in regions with high incidence of cervical cancer.

OBJECTIVE: To compare papillomavirus types found in histologically rare cervical carcinomas (n = 29) with those types found in common cervical carcinomas (n = 14) archived at the Institute of Cancer in Mexico City, Mexico.

DESIGN: Paraffin-embedded tissues were received and sectioned at the Louisiana State University Health Sciences Center at Shreveport. One section for each block was stained and examined by 2 pathologists. Specific histologies were categorized into 2 broad groups: common (squamous cell carcinoma or adenocarcinoma) or rare (adenosquamous, papillary, villoglandular, anaplastic, transitional, spindle, adenoid basal, colloid, neuroendocrine, and glassy cell carcinomas). Papillomavirus typing results were based on Roche Molecular Systems line-blot assay.

RESULTS: No significant difference was found for dual HPV types (21% of both groups), positivity for HPV-16 (66% of rare tumors and 71% of common tumors), or absence of HPV types 16 or 18, although the rare cancers had a greater tendency toward more unusual HPV types (8/29 rare tumors and 1/14 common tumors had no HPV- 16 or HPV-18 DNA). Non-HPV-16/18 types found only in rare tumors included HPV types 52, 84, 26, 35, and 58.

CONCLUSIONS: Rare types of cervical carcinoma also are associated with papillomavirus, most with types similar to those found in common cervical neoplasias.


Determinants for genital human papillomavirus (HPV) infection in 1000 randomly chosen young Danish women with normal Pap smear: are there different risk profiles for oncogenic and nononcogenic HPV types?

Kjaer SK, van den Brule AJ, Bock JE, Poll PA, Engholm G, Sherman ME, Walboomers JM, Meijer CJ.

Danish Cancer Society, Division for Cancer Epidemiology, Copenhagen, Denmark.

Cancer Epidemiol Biomarkers Prev 1997 Oct;6(10):799-805 Abstract quote

Most studies of risk factors for human papillomavirus (HPV) DNA detection have focused on overall HPV positivity and have not examined determinants for high-risk and low-risk HPV types separately.

We studied risk determinants for genital HPV infection in 1000 randomly chosen women (20-29 years) with normal cervical cytology from Copenhagen, Denmark. All women had a personal interview, a Pap smear, and cervical swabs for HPV DNA detection using a PCR technique. On the basis of their association with cervical cancer, the HPV types were categorized as belonging to a high-risk group ("oncogenic types") or a low-risk group ("nononcogenic types"). The overall HPV detection rate was 15.4%. Of HPV-positive women, 74% had oncogenic HPV types, and 30% had nononcogenic HPV types.

Younger age and lifetime measures of sexual activity (notably, number of partners) were the main risk factors for the oncogenic HPV types. Furthermore, a previous Chlamydia infection was associated with the high-risk HPV types.

In contrast, the most important determinants for nononcogenic HPV infection were contraceptive variables related to the physical protection of the cervix (condom or diaphragm) and number of partners in the last 4 or 12 months. Our study confirms the venereal nature of HPV infection.

We hypothesize that the low-risk HPV infection, which correlates with recent sexual behavior, may be more transient than infection with the oncogenic HPV types, which correlates with lifetime exposure measurements of sexual habits.

Human Papillomavirus–16 E6 Variants in Cervical Squamous Intraepithelial Lesions From HIV-Negative and HIV-Positive Women

Lucía Pérez-Gallego, MD, PhD, Gema Moreno-Bueno, PhD, David Sarrió, PhD, Asunción Suárez, MD, Carlos Gamallo, MD, PhD, and José Palacios, MD, PhD

Am J Clin Pathol 2001;116:143-148 Abstract quote

We studied 48 human papillomavirus (HPV)-16–positive squamous intraepithelial lesions (SILs) from HIV-negative patients (16 low-grade SILs [LSILs]; 32 high-grade SILs [HSILs]) and 13 HPV-16–positive SILs from HIV-positive patients with AIDS (1 LSIL; 12 HSILs). After HPV typing, the entire HPV-16 E6 coding region was amplified and sequenced in all samples. We detected 12 HPV-16 E6 prototypes and 4 variants among the LSILs in HIV-negative patients, and 15 HPV-16 E6 prototypes and 17 HPV-16 variants in the HSIL group. The most prevalent variant of SIL types was European 350G, present in 3 and 13 cases, respectively. In 3 HSILs and no LSILs we found mixed infection by an HPV-16 E6 prototype and a variant. Two variants (1 each in LSIL and HSIL) were of non-European lineage. The only LSIL in HIV-positive patients had an HPV-16 E6 prototype; in the HSILs, we found 8 HPV-16 E6-prototypes, 4 with mixed infection with HPV-31 and 4 variants, all European 350G.

The higher proportion of HPV-16 E6 variants in HSIL than in LSIL in HIV-negative patients suggests a greater risk of progression. However, further studies are needed.

A prospective study of human papillomavirus (HPV) type 16 DNA detection by polymerase chain reaction and its association with acquisition and persistence of other HPV types.

Liaw KL, Hildesheim A, Burk RD, Gravitt P, Wacholder S, Manos MM, Scott DR, Sherman ME, Kurman RJ, Glass AG, Anderson SM, Schiffman M.

Department of Epidemiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.

J Infect Dis 2001 Jan 1;183(1):8-15 Abstract quote

Human papillomavirus (HPV)-16 causes about half the cases of cervical cancer worldwide and is the focus of HPV vaccine development efforts. Systematic data are lacking as to whether the prevention of HPV-16 could affect the equilibrium of infection with other HPV types and thus alter the predicted impact of vaccination on the occurrence of cervical neoplasia.

Therefore, the associations of HPV-16 detection with subsequent acquisition of other HPV types and with the persistence of concomitantly detected HPV types were examined prospectively among 1124 initially cytologically normal women. Preexisting HPV-16 was generally associated with an increased risk for subsequent acquisition of other types. HPV-16 did not affect the persistence of concomitant infections, regardless of type.

These findings suggest that the prevention or removal of HPV-16 is not likely to promote the risk of infection with other types, a theoretical concern with current vaccination efforts.

Penile lesions and human papillomavirus in male sexual partners of women with cervical intraepithelial neoplasia.

Bleeker MC, Hogewoning CJ, Van Den Brule AJ, Voorhorst FJ, Van Andel RE, Risse EK, Starink TM, Meijer CJ.

Department of Pathology, Section Molecular Pathology, VU University Medical Center, Amsterdam, The Netherlands.

J Am Acad Dermatol 2002 Sep;47(3):351-7 Abstract quote

BACKGROUND: Genital human papillomavirus infection (HPV) is causally associated with cervical carcinomas and premalignant lesions. Limited information is available about the prevalence of HPV and penile lesions in male sexual partners of women with cervical intraepithelial neoplasia (CIN).

OBJECTIVE: The aim of this study was to identify the presence of penile lesions and HPV in penile scrapings from male sexual partners of women with CIN.

METHODS: One hundred seventy-five male sexual partners of women with CIN were screened by peniscopy after acetowhite staining and HPV testing on penile scrapings.

RESULTS: Penile lesions were seen in 68% of the male sexual partners. More than one lesion type was diagnosed in 15%. Flat lesions, papular lesions, and condylomata acuminata were seen in 83%, 29%, and 4%, respectively. HPV was detected in 59% of the penile scrapings, containing mainly oncogenic HPV types. When penile lesions were present at peniscopy, 67% of penile scrapings were positive for HPV, whereas 37% were HPV-positive when no lesions were visible.

CONCLUSIONS: Penile lesions are frequently found in sexual partners of women with CIN. Most of these lesions are subclinical (ie, only visible after acetowhite staining) and are often associated with the presence of high-risk HPV, indicating that male sexual partners of women with CIN might constitute a reservoir for high-risk HPV.

SMOKING Am J Epidemiol 1988;128:74-77


Chlamydia trachomatis serotypes

JAMA 2001 285:47-51

Longitudinal nested case control study of cohort of 530,000 women in Finland, Norway, and Sweeden
Serum studies measured at least 12 months before development of carcinoma
Serotype G was most strongly associated with squamous cell carcinoma

C. trachomatis DNA was found in 5% of cases of CIN


Treatment for cervical intraepithelial neoplasia and risk of preterm delivery.

Sadler L, Saftlas A, Wang W, Exeter M, Whittaker J, McCowan L.

Department of Obstetrics and Gynaecology, University of Auckland, National Women's Hospital, Auckland, New Zealand
JAMA. 2004 May 5;291(17):2100-6. Abstract quote  

CONTEXT: It is unclear whether treatments for cervical intraepithelial neoplasia (CIN) increase the subsequent risk of preterm delivery. Most studies have lacked sufficient sample size, mixed heterogeneous subtypes of preterm delivery, and failed to control for confounding factors.

OBJECTIVE: To determine whether cervical laser and loop electrosurgical excision procedure (LEEP) treatments increase risk of preterm delivery and its subtypes.

DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study conducted among women evaluated at a colposcopy clinic serving Auckland, New Zealand (1988-2000), comparing delivery outcomes of untreated women (n = 426) and those treated (n = 652) with laser conization, laser ablation, or LEEP. Record linkage using unique health identifiers identified women who had subsequent deliveries.

MAIN OUTCOME MEASURES: Total preterm delivery and its subtypes, spontaneous labor and premature rupture of membranes before 37 weeks' gestation (pPROM).

RESULTS: The overall rate of preterm delivery was 13.8%. The rate of pPROM was 6.2% and the rate of spontaneous preterm delivery was 3.8%. Analyses showed no significant increase in risk of total preterm delivery (adjusted relative risk [aRR], 1.1; 95% confidence interval [CI], 0.8-1.5) or spontaneous preterm delivery (aRR, 1.3; 95% CI, 0.7-2.6) for any treatment. Risk of pPROM was significantly increased following treatment with laser conization (aRR, 2.7; 95% CI, 1.3-5.6) or LEEP (aRR, 1.9; 95% CI, 1.0-3.8), but not laser ablation (aRR, 1.1; 95% CI, 0.5-2.4). Moreover, risk of pPROM and total preterm delivery increased significantly with increasing height of tissue removed from the cervix in conization. Women in the highest tertile of cone height (> or =1.7 cm) had a greater than 3-fold increase in risk of pPROM compared with untreated women (aRR, 3.6; 95% CI, 1.8-7.5).

CONCLUSIONS: LEEP and laser cone treatments were associated with significantly increased risk of pPROM. Careful consideration should be given to treatment of CIN in women of reproductive age, especially when treatment might reasonably be delayed or targeted to high-risk cases.


Quantitation of CDC6 and MCM5 mRNA in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix.

Murphy N, Ring M, Heffron CC, Martin CM, McGuinness E, Sheils O, O'leary JJ.

[1] 1Department of Pathology, Coombe Women's Hospital, Dublin, Ireland [2] 3Trinity College Dublin, Dublin, Ireland.

Mod Pathol. 2005 Jun;18(6):844-9. Abstract quote  

CDC6 and MCM5 play essential roles in eukaryotic DNA replication. Several studies have highlighted the potential of these proteins as molecular markers of dysplastic and malignant cells in histopathological diagnosis. The mode of expression of CDC6 and MCM5 mRNA and their significance in normal, dysplastic and malignant cervical cells remains to be elucidated.

Using a quantitative real-time RT PCR assay, we compared CDC6 and MCM5 mRNA expression in normal cervical epithelium, cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix. Our study cohort comprised 20 normal cervical biopsies, 20 CIN3 and eight invasive squamous cell carcinomas. All samples were formalin fixed and paraffin embedded. Total RNA was extracted and analysed for expression of GAPDH, CDC6 and MCM5 using real-time quantitative TaqMan RT-PCR.

A linear increase in MCM5 and CDC6 mRNA expression is observed in normal cervix, CIN3 and invasive cervical carcinoma. The overall difference in MCM5 mRNA expression in the normal cervix, CIN3 and invasive cohort groups is highly statistically significant (P=0.001). An increase in CDC6 mRNA expression in CIN3 and invasive cervical squamous cell carcinoma was observed; however, the overall difference between cohort groups was not found to be statistically significant (P=0.104). Increased transcription of MCM5 and CDC6 occurs as a consequence of cervical neoplastic progression. This pattern of increased mRNA expression in CIN3 and invasive cervical carcinoma directly correlates with findings at the phenotypic protein expression level.

This study further confirms the importance of MCM5 and CDC6 in malignant transformation and in the pathogenesis of cervical dysplasia.

Immunohistochemical localization of cdc6 in squamous and glandular neoplasia of the uterine cervix.

Bonds L, Baker P, Gup C, Shroyer KR.

Department of Pathology, School of Medicine, University of Colorado Health Sciences Center, Denver.

Arch Pathol Lab Med 2002 Oct;126(10):1164-8 Abstract quote

Context.-Cdc6 has been extensively studied as a marker for cellular proliferation that is expressed during the normal cell cycle. Recent studies indicate that Cdc6 may be a marker for cervical intraepithelial neoplasia (CIN) and carcinoma; however, the histologic distribution of Cdc6 has not been explicitly defined. Expression of Cdc6 in the endocervical mucosa also remains unexplored.

Objective.-The goal of the current study was to evaluate the distribution of Cdc6 protein, MIB-1 protein, and human papillomavirus (HPV) DNA in a broad range of cervical tissues, including normal, potentially premalignant, and malignant lesions of the ectocervical and endocervical mucosa.

Methods.-We used an indirect immunoperoxidase method to stain formalin-fixed, paraffin-embedded tissues and frozen tissues, including biopsy and hysterectomy specimens, for Cdc6 and MIB-1 proteins, and we used in situ hybridization to detect HPV DNA in a subset of cases.

Results.-Cdc6 staining was exclusively nuclear and was present in both squamous and glandular epithelial cells of histologic sections. Cdc6 staining was rarely present in specimens of normal cervical squamous mucosa (2/84, 2.4%) or in specimens with squamous metaplasia (3/59, 5.1%) and was not detected in normal endocervical glands (0/84). Staining was present in most cases of CIN I (31/48, 65%). Staining was present in the majority of cases of CIN II (25/28, 89%) and in all cases of CIN III (36/36) and squamous cell carcinomas (34/34). The proportion of cells staining for Cdc6 increased with the grade of dysplasia, and the proportion of stained cells in squamous cell carcinomas was similar to that in lesions of high-grade dysplasia. Cdc6 staining was present in the majority of cases in glandular lesions including adenocarcinoma in situ (11/14, 79%) and adenocarcinoma (8/10, 80%). The histologic distribution of Cdc6-immunoreactive cells was similar to that of cells with a strong signal for HPV DNA, but Cdc6 protein and HPV DNA did not colocalize at the level of individual cells.

Conclusion.-Cdc6 expression is a marker for high-grade cervical squamous and glandular dysplasia and carcinoma and is associated with HPV infection. The mechanistic basis of the association between HPV infection and Cdc6 immunopositivity remains to be determined but may represent either up-regulation of Cdc6 expression or stabilization of the Cdc6 protein.

Immunohistochemical and ultrastructural features of Langerhans cells in condyloma acuminatum.

Department of Dermatology, The Second Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China.


J Cutan Pathol. 2008 Jan;35(1):15-20. Abstract quote

Background: There are few studies on the abnormal morphology of Langerhans cells (LCs) in condyloma acuminatum (CA) lesions and the essence of the abnormal morphology of LCs in CA lesions is still not well elucidated. The aim of this study was to further investigate the morphological features of LCs in CA lesions.

Methods: CD1a(+) LCs in 13 CA lesions and in 13 normal controls were labeled using immunohistochemistry and examined by light microscopy. Ultrastructural investigation on LCs in six CA lesions and in six normal controls was performed by electron microscopy.

Results: Compared with those in normal controls, most CD1a(+) LCs in CA lesions exhibited dysplastic dendrities and abnormal distribution. The number of CD1a(+) LCs in CA lesions (26.31 +/- 18.84) was statistically lower (p < 0.001) than that in normal controls (72.00 +/- 27.40). Electron microscopy showed that the number of Birbeck granules within lesional LCs (4.00 +/- 2.94) was significantly decreased (p < 0.001) than that within normal LCs (10.80 +/- 4.78). The ultrastructures of most lesional LCs displayed degenerative changes.

Conclusions: The morphology of most LCs in CA lesions shows degenerative changes, which suggest that these LCs have been functionally impaired.
Maspin expression in CIN 3, microinvasive squamous cell carcinoma, and invasive squamous cell carcinoma of the uterine cervix.

Xu C, Ruhul Quddus M, James Sung C, Steinhoff MM, Zhang C, Dwayne Lawrence W.

1Department of Pathology, Women and Infants Hospital and Brown Medical School, Providence, RI, USA.

Mod Pathol. 2005 Aug;18(8):1102-6. Abstract quote  

Maspin is a serine protease inhibitor with tumor suppression activity. It is expressed in normal breast and prostate tissue but is downregulated or absent in breast and prostate tumors. Recent reports have shown that decreased expression is associated with a greater propensity for metastasis in oral squamous cell carcinomas.

We know that some high-grade cervical intraepithelial neoplasia progress to invasive carcinomas while others either persist at the same degree of atypia or regress.

The pattern of maspin expression in cervical intraepithelial neoplasia-grade 3, microinvasive squamous carcinomas and overtly invasive squamous cell carcinomas of the uterine cervix was studied to determine the relationship between the extent of maspin expression and the progression of cervical intraepithelial neoplasia to squamous cell carcinoma. In total, 36 cases were evaluated: 18 cases of cervical intraepithelial neoplasia-grade 3, seven cases of microinvasive squamous cell carcinoma and 11 cases of invasive squamous cell carcinoma.

A monoclonal antibody was used on paraffin-embedded tissues. Immunoreactivity was scored semiquantitatively using a scale of 0-3. The sums of the scores of the different groups were compared using the Mann-Whitney U-test. A significant decrease in maspin scores was noted between cervical intraepithelial neoplasia-grade 3 vs invasive squamous cell carcinoma (P<0.005), microinvasive squamous cell carcinoma vs invasive squamous cell carcinoma (P<0.05), and cervical intraepithelial neoplasia-grade 3 vs tumor emboli (P<0.005). Although not statistically significant, scores of cervical intraepithelial neoplasia-grade 3 associated with invasive squamous cell carcinoma were lower compared to that cervical intraepithelial neoplasia-grade 3 without invasive squamous cell carcinoma.

These findings suggest that maspin likely plays a role in disease progression from in situ to invasive carcinoma. Virtual absence of maspin immunopositivity in tumor emboli indicates that maspin may also play a role in metastasis.

MUC4 expression is increased in dysplastic cervical disorders

Anna López-Ferrer, MS
Francesc Alameda, MD, PhD
Carlos Barranco, MD, PhD
Marta Garrido, TS
Carme De Bolós, PhD

Hum Pathol 2001;32:1197-1202. Abstract quote

The female uterine cervix has 2 characteristic populations of epithelial cells: the endocervix is composed by mucus-secreting cells that express several mucin genes, and the exocervix has a typical stratified squamous epithelium and does not express secreted mucins. Among human mucin genes, the MUC4 sequence has a transmembrane domain, and its molecular structure suggests that it has a protective role and also may be implicated in intracellular signalling.

The aim of this study is to analyze whether changes in the expression of MUC4 can be detected associated with the squamous dysplastic transformation of exocervical epithelium. MUC4 expression has been analyzed by immunohistochemistry, Western blotting, and in situ hybridization.

Using immunohistochemical techniques, MUC4 is found in normal endocervix (n = 11) and is absent or only focally detected in the normal stratified cervical epithelium (n = 18). In samples from squamous metaplasia (n = 9), MUC4 is variably expressed (10% to 50% positive cells), whereas MUC4 is strongly detected in dysplastic cervical epithelia. The greatest number of positive cells is found in samples with moderate and severe dysplasia in which MUC4 is detected in 100% of the analyzed samples (n = 16). These results have been confirmed by Western blotting and by detection of MUC4 transcripts using in situ hybridization.

The present data suggest that MUC4 is activated during the process of squamous dysplastic transformation and may be used as a marker for this pathologic process.

Survivin as a Marker of Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus and a Predictor of Virus Clearance and Prognosis in Cervical Cancer

Margherita Branca, MD, PhD, MIAC, etal.
Am J Clin Pathol 2005;124:113-121 Abstract quote

We analyzed survivin as a marker of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HR-HPV) and a predictor of HPV clearance and disease outcome in cervical cancer in 302 samples (squamous cell carcinomas [SCCs], 150; CIN lesions, 152) by immunohistochemical staining with survivin antibody and HPV testing using polymerase chain reaction.

HR-HPV types were associated closely with CIN and SCC. There was a significant linear relationship between grade and intensity of survivin expression (P = .0001). Survivin overexpression also was associated strongly with HR-HPV type (P = .0001). Multivariate regression analysis revealed survivin and p16INK4a as equally strong independent predictors of HR-HPV. Deregulated survivin expression did not predict clearance or persistence of HR-HPV after treatment of CIN or survival in cervical cancer in univariate (P = .417) or multivariate analysis. After adjustment for HR-HPV, stage, age, and tumor grade in the Cox regression model, only stage (P = .0001) and age (P = .0001) remained independent prognostic predictors.

Survivin seems to be an early marker of cervical carcinogenesis. Up-regulated survivin expression was an independent predictor of HR-HPV in cervical lesions, most plausibly explained by its normal transcriptional repression by wild-type p53 being eliminated by HR-HPV E6 oncoprotein.
Immunohistochemical Localization of Survivin in Benign Cervical Mucosa, Cervical Dysplasia, and Invasive Squamous Cell Carcinoma

Michael Frost, MBBS
Elke A. Jarboe, MS
David Orlicky, PhD
Roberto Gianani, MD
L. Chesney Thompson, MD
Takayuki Enomoto, MD, PhD
Kenneth R. Shroyer, MD, PhD

Am J Clin Pathol 2002;117:738-744 Abstract quote

Survivin is an inhibitor of apoptosis protein (IAP) that is expressed in fetal development and in cancer. Survivin expression in premalignant lesions remains undefined.

We obtained 73 samples of cervical squamous tissue, including 31 normal, 17 low- and 15 high-grade squamous intraepithelial lesions (LSILs, HSILs), and 10 squamous cell carcinomas (SCCs) from cone biopsy and hysterectomy specimens, and stained for survivin using an immunoperoxidase method. Nuclear staining was detected in normal mucosa, LSILs, and HSILs; staining intensity was greatest in cases with morphologic evidence of human papillomavirus (HPV) infection. In situ hybridization of serial sections demonstrated colocalization of HPV DNA and survivin. Cytoplasmic staining was observed in immature squamous metaplasia and in SCCs.

Survivin expression in immature metaplastic squamous mucosa may reflect a role for survivin in normal squamous differentiation. However, the histologic correlation between nuclear staining and HPV infection suggests involvement of survivin in HPV-mediated disruption of normal cellular maturation.


Transforming growth factor-beta1 induces tumor stroma and reduces tumor infiltrate in cervical cancer.

Hazelbag S, Gorter A, Kenter GG, Van Den Broek L, Fleuren G.

Department of Pathology and Department of Obstetrics and Gynecology, Leiden University Medical Center, Leiden, The Netherlands.


Hum Pathol 2002 Dec;33(12):1193-9 Abstract quote

Cervical carcinomas consist of tumor cell nests surrounded by varying amounts of intratumoral stroma containing different quantities and types of immune cells. Besides controlling (epithelial) cell growth, the multifunctional cytokine transforming growth factor-beta(1) (TGF-beta(1)) is involved in the formation of stroma and extracellular matrix (ECM) and in immunosuppression.

Several malignancies are known to be associated with enhanced production of TGF-beta(1), repression or mutation of TGF-beta transmembrane receptors, or mutations at the postreceptor intracellular signaling pathway.

The aim of our study was to investigate the role of tumor cell-derived TGF-beta(1) on the amount of intratumoral stroma; the deposition of collagen IV, fibronectin, and laminin; and the tumor infiltrate in cervical carcinoma.

The expression of TGF-beta(1) mRNA in 108 paraffin-embedded cervical carcinomas was detected by mRNA in situ hybridization. Immunohistochemistry was used to investigate the amount of tumor stroma and ECM proteins and the extent of the tumor infiltrate. Plasminogen activator inhibitor-1 (PAI-1) protein expression in tumor cells was determined to verify the biological activity of TGF-beta(1.) Cytoplasmatic TGF-beta(1) mRNA expression in tumor cells was significantly correlated with the amount of intratumoral stroma and the deposition of collagen IV. TGF-beta(1) mRNA expression in every tumor was accompanied by PAI-1 expression, indicating biological activity of TGF-beta(1). An inverse relationship between TGF-beta(1) mRNA expression in tumor cells and the extent of the tumor infiltrate was demonstrated.

Our results indicate that cervical cancer cells affect the amount and the composition of the intratumoral stroma and the tumor infiltrate by the production and secretion of TGF-beta(1).




Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia.

Fujii T, Masumoto N, Saito M, Hirao N, Niimi S, Mukai M, Ono A, Hayashi S, Kubushiro K, Sakai E, Tsukazaki K, Nozawa S.

Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo 160-8582, Japan.
Diagn Mol Pathol. 2005 Jun;14(2):103-8. Related Articles, Links

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization.

We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome.

We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.


Human Papillomavirus (HPV) Profiles of Vulvar Lesions: Possible Implications for the Classification of Vulvar Squamous Cell Carcinoma Precursors and for the Efficacy of Prophylactic HPV Vaccination.

Departments of *Pathology and Molecular Medicine ‡Obstetrics and Gynecology, Charles University, 2nd Medical School, Faculty Hospital Motol †Department of Experimental Virology, National Reference Laboratory for Papillomaviruses, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.


Am J Surg Pathol. 2007 Dec;31(12):1834-1843. Abstract quote

The term vulvar intraepithelial neoplasia (VIN) introduced in 1986 incorporates 3 grades of usual VIN (u-VIN I-III) and the differentiated VIN (d-VIN). Although u-VIN is etiologically associated with the human papillomavirus (HPV) infection, d-VIN represents an alternative HPV negative pathway of vulvar carcinogenesis. In 2004, the u-VIN I category was abandoned and u-VIN II and III were merged. Further, an alternative Bethesda-like terminology scheme presenting the term vulvar intraepithelial lesion was proposed recently.

To analyze the impact of HPV profiles of vulvar precancerous lesions for their classification and to assess the presumable efficacy of the prophylactic HPV vaccination, 269 vulvar excisions representing lichen sclerosus, lichen simplex chronicus, condylomata acuminata, d-VIN, all grades of u-VIN and squamous cell carcinomas were subjected to the HPV typing by use of GP5+/6+ polymerase chain reaction and reverse line blot hybridization. The results showed different HPV profiles, and also differing frequency of multiple-type HPV infection and the age structure in patients with u-VIN II and III. The biologic heterogeneity within the u-VIN II category was also demonstrated. u-VIN I was distinguished as a rare disorder associated with high-risk HPV infection.

We conclude that the original VIN terminology proposed in 1986 seems to be appropriate for the classification of vulvar squamous dysplastic lesions. The spectrum of HPV types found in vulvar squamous cell carcinomas indicates that the efficacy of HPV vaccination in preventing vulvar cancer might be diminished in the studied population, because the recently developed prophylactic vaccines are targeted against a limited number of HPV types.
Cervical adenoid basal tumors comprised of adenoid basal epithelioma associated with various types of invasive carcinoma: Clinicopathologic features, human papillomavirus DNA detection, and P16 expression.

Parwani AV, Smith Sehdev AE, Kurman RJ, Ronnett BM.
Hum Pathol. 2005 Jan;36(1):82-90 Abstract quote.  

Summary Adenoid basal tumors are uncommon cervical lesions that some pathologists consider invasive carcinomas but others consider "epitheliomas" due to their low-grade histological appearance and rarely documented malignant behavior.

We report the clinicopathologic features of 10 tumors comprised of both typical low-grade adenoid basal tumors (epitheliomas) intimately associated with invasive carcinomas having infiltrative growth, increased cytological atypia and mitotic activity, and various types of differentiation, including adenoid basal/squamous, pure squamous, adenoid cystic, and small cell neuroendocrine. Tumors were evaluated for the presence of human papillomavirus (HPV) DNA and immunohistochemical p16 expression. The patients in the study group ranged in age from 45 to 81 years (mean, 65 years). Most of the patients presented with abnormal cervicovaginal smears. The initial diagnosis was made on specimens obtained by cervical biopsy, laser electrocautery excision procedure (LEEP), or cone biopsy in 8 patients. Two 2 patients were incidentally diagnosed in hysterectomy specimens. All 10 patients had squamous intraepithelial lesions (9 high-grade, 1 low-grade). In all cases diagnosed in LEEP or cone biopsy specimens, the invasive carcinoma component was present in the excisional specimen and extended to the margins. Seven patients diagnosed on excisional or biopsy specimens who underwent hysterectomy had residual tumor in the cervix, ranging from microscopic foci to deeply invasive. No lymph node metastases were identified in 4 patients who were staged. Seven patients with follow-up were alive without evidence of disease after follow-up intervals of 8 to 84 months (mean, 45 months; median, 29 months). One patient with a component of small cell carcinoma died of other causes without evidence of disease at 18 months. HPV 16 DNA was detected in both the adenoid basal epithelioma and invasive carcinoma components in 9 tumors by in situ hybridization, and HPV 33 was detected by polymerase chain reaction in 1 tumor. All tumors expressed p16 diffusely.

Adenoid basal tumors are high-risk HPV-related tumors that can be comprised of both a low-grade adenoid basal tumor, which can be designated as epithelioma, and invasive carcinomas of various types. The invasive component is usually evident in the excisional biopsy specimen, allowing for recognition of a tumor that needs further management.

A reappraisal of "Basaloid Carcinoma" of the cervix, and the differential diagnosis of basaloid cervical neoplasms.

Grayson W, Cooper K.

Adv Anat Pathol 2002 Sep;9(5):290-300 Abstract quote

"Basaloid carcinoma" of the uterine cervix is a neglected and underrecognized entity that is not included in the current World Health Organization's classification of cervical neoplasms. Historically, this term has been used synonymously with adenoid basal carcinoma (ABC). In recent years, however, it has become evident that a broad spectrum of basaloid cervical neoplasms exist. At one end of the spectrum are low-grade lesions, such as ABC; at the opposite end of the spectrum there are aggressive tumors, including adenoid cystic carcinoma, large cell neuroendocrine carcinoma, and basaloid squamous carcinoma.

The purpose of this review is to revisit the concept of basaloid tumors of the cervix, to define their morphologic spectrum, and to address potential pitfalls in the differential diagnosis. To avoid confusion, use of the term "basaloid squamous cell carcinoma" is recommended when diagnosing a cervical tumor with histologic features of "basaloid carcinoma," as seen in other anatomic sites. A proposed classification of basaloid tumors of the uterine cervix is also presented.

Extensively keratinized squamous intraepithelial lesions

Am J Clin Pathol 2001;115:80-84

These are difficult to grade with poor interobserver agreement

Should be diagnosed as "SIL, grade cannot be determined due to extensive keratinization"

KEY Features:
Elongate or caudate forms
Cytoplasmic hypereosinophilia and/or orangeophilia
Opaque and/or pyknotic nuclei

Eosinophilic Dysplasia of the Cervix: A Newly Recognized Variant of Cervical Squamous Intraepithelial Neoplasia.

Ma L, Fisk JM, Zhang RR, Ulukus EC, Crum CP, Zheng W.

From the Departments of *Pathology and double daggerObstetrics and Gynecology, Yale University School of Medicine, New Haven, CT; and daggerDepartment of Pathology, Brigham and Women's Hospital, Harvard University, Boston, MA.

Am J Surg Pathol. 2004 Nov;28(11):1474-1484. Abstract quote  

A proportion of cervical squamous intraepithelial lesions encountered in surgical pathology practice contain both metaplastic features and some degree of atypia [so-called eosinophilic dysplasia (ED)] that defy classification according to established criteria.

To elucidate the nature of these lesions, we compared 44 cases of ED to 20 classic high-grade squamous intraepithelial lesions (HSILs) and 10 squamous metaplasias using a panel of biomarkers and human papillomavirus (HPV) testing. EDs were defined as 1) lack of normal maturation; 2) relatively abundant eosinophilic cytoplasm and distinct cell borders compared with conventional HSIL; 3) mildly to moderately increased nuclear to cytoplasmic ratio; and 4) focal dysplastic nuclei showing nuclear enlargement, hyperchromasia, variable nuclear membrane irregularities, and appreciable nucleoli. Expression of p16 (p16), MIB-1 (Ki-67) labeling index, and HPV DNA detection and typing were performed on each case. The majority of EDs showed more than three atypical cells in an entire lesion but lack of apparent features of HSIL. It was common to find neighboring cervical squamous metaplasia and/or conventional SILs (either HSIL or low-grade squamous intraepithelial lesion [LSIL]). Among the 44 cases, 18 (45%) ED lesions were found to be associated with HSIL, 15 (34%) with LSIL or condylomatous lesions, and 13 (30%) EDs were seen without any SILs in the entire specimens. Area of benign squamous metaplasia was found in all ED cases. High levels of p16 and MIB-1 expression were seen in 41 (93%) of 44 ED cases with degrees of immunoreactivity closely resembling those seen with HSIL. Of 16 EDs tested, 13 (81%) were positive for HPV DNAs. Among 10 HPV-positive cases subtyped, 9 (90%) cases contained intermediate- and/or high-risk HPVs and 1 case contained a novel HPV.

In the follow-up of pure ED cases, the majority showed presence of dysplastic lesions of either HSIL or LSIL on either loop electric excision procedures or Papanicolaou test samples after a 6- to 10-week period. Therefore, ED represents an unrecognized and potentially clinically significant subgroup of cervical intraepithelial lesions. Based on the unique histologic appearance of ED, its association in some cases with HSIL, the overall immunohistochemical findings, frequent association of ED with intermediate- and/or high-risk HPV infection, and limited follow-up data, we believe that ED represents a variant of HSIL (CIN 2).

Since ED possesses histologic features of both dysplasia and metaplasia, we speculate that it may arise from metaplastic cervical squamous epithelium that has subsequently become infected with intermediate- or high-risk HPV.
Eosinophils as a Marker for Invasion in Cervical Squamous Neoplastic Lesions

Gregory W Spiegel, M.D., Ph.D.; Mubina Ashraf, M.D.; John J. S. Brooks, M.D.

From the Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York.

Int J Gyn Pathol 2002;21:117-124 Abstract quote

A study of eosinophils associated with cervical neoplastic squamous epithelium was undertaken to determine whether their presence is a marker for invasion.

Forty cervical incisional biopsy specimens of high-grade squamous intraepithelial neoplasia (HSIL), 12 with an element of invasive carcinoma, and 2 of HSIL suspicious for invasion, and follow-up excisional specimens of 27 cases of HSIL and 6 of microinvasive and invasive carcinoma were reviewed. In both incisional biopsy and excisional specimens, the presence of 5 eosinophils/high-power fields (hpf) and 10 eosinophils/10 hpf were both highly significantly associated with invasion with a high degree of specificity and positive predictive value, whereas counts below these thresholds had a high negative predictive value.

The authors propose: 1) eosinophil counts in cervical incisional biopsy specimens of 5/hpf and/or 10/10 hpf warrant a note of caution that invasion may be present even when none is identified in the specimen by conventional criteria; 2) eosinophil counts of 5/hpf and/or 10/10 hpf in excisional biopsy and hysterectomy specimens should raise the suspicion of invasion in cases in which only HSIL is identified in the initial sections, and warrant additional sections and/or levels to search for invasion; and 3) the above eosinophil counts may provide supportive evidence for invasion in cases with equivocal invasion by conventional criteria.

Glassy Cell Carcinoma of the Uterine Cervix: Histochemical, Immunohistochemical, and Molecular Genetic Observations

Noriko Kato, M.D.; Yousei Katayama, M.D.; Mitsuomi Kaimori, M.D.; Teiichi Motoyama, M.D.

From the Department of Pathology (N.K., T.M.), Yamagata University School of Medicine, Yamagata; the Department of Pathology (Y.K.), Hachinohe Municipal Hospital, Hachinohe; and the Department of Pathology (M.K.), Aomori Central Hospital, Aomori, Japan.

Int J Gynecol Pathol 2002;21:134-140 Abstract quote

Glassy cell carcinoma (GCC) of the uterine cervix is characterized by distinctive cytological features and an aggressive clinical course. Although this tumor has been usually considered a poorly differentiated variety of adenosquamous carcinoma (ASC), a clarification of the phenotype and histogenesis of GCC is still required.

We examined three GCCs and four ASCs for histochemical and immunohistochemical phenotypes and molecular genetic status, comparing them with five nonkeratinizing squamous cell carcinomas and five endocervical-type mucinous adenocarcinomas. GCCs had a profile of cytokeratin expression similar to that of reserve cells or immature squamous cells of the cervix. In addition to squamous differentiation, GCCs sporadically produced intestinal-type mucin. HPV 18 was detected in two of three GCCs and two of four ASCs.

GCCs may originate from multipotential stem or reserve cells that undergo early squamous differentiation. The presence of HPV 18 might stimulate biphasic squamous and glandular differentiation.


Highly Differentiated Keratinizing Squamous Cell Cancer of the Cervix A Rare, Locally Aggressive Tumor Not Associated With Human Papillomavirus or Squamous Intraepithelial Lesions

Carl Morrison, M.D., D.V.M.; Francesco Catania, M.D.; Paul Wakely, Jr., M.D.; Gerard J. Nuovo, M.D.

From the Departments of Pathology (C.M., P.W., G.J.N.) and Obstetrics and Gynecology (F.C.), Ohio State University Medical Center, Columbus, Ohio, U.S.A.

Am J Surg Pathol 2001;25:1310-1315 Abstract quote

The purpose of this study is to report an unusual variant of cervical squamous cell carcinoma, not associated with either human papillomavirus infection or antecedent squamous intraepithelial lesions.

Five women had a diagnosis of invasive cervical cancer discovered at hysterectomy performed for prolapse (two cases), leiomyoma (one case), or a vaginal fistula (two cases). The women ranged in age from 47 to 78 years (mean 59 years). Four of the five had a history of normal Papanicolaou (Pap) smears; the other had a Pap smear diagnosis of atypical squamous cells of undetermined significance (ASCUS). All had large cervical tumors (two with parametrial involvement and one with vaginal involvement) that showed extensive keratin formation, an inverted pattern of growth, and, except for one case, minimal cytologic atypia. There was extensive hyperkeratosis and parakeratosis adjacent to each tumor; none had evidence of squamous intraepithelial lesion. Human papillomavirus testing by polymerase chain reaction in situ hybridization and reverse-transcribed polymerase chain reaction in situ was negative in each case, compared with a detection rate of 107 of 108 (99%) for squamous intraepithelial lesion-associated cervical squamous cell and adenocarcinomas. Two of the women died of extensive local recurrence; two other women were recently diagnosed.

We conclude that highly differentiated keratinizing squamous cell carcinoma of the cervix is a rare entity not associated with human papillomavirus infection or squamous intraepithelial lesion and thus difficult to detect on routine cervical cancer screening.

Detection of microinvasion in vulvar and cervical intraepithelial neoplasia using double immunostaining for cytokeratin and basement membrane components.

Rush D, Hyjek E, Baergen RN, Ellenson LH, Pirog EC.

Department of Pathology and Laboratory Medicine, Malcolm Randall VA Medical Center, and University of Florida College of Medicine, Gainesville, USA.

Arch Pathol Lab Med. 2005 Jun;129(6):747-53. Abstract quote  

CONTEXT: Identification of early invasion in vulvar intraepithelial neoplasia 3 (VIN 3) and cervical intraepithelial neoplasia 3 (CIN 3) may be difficult with the use of routine hematoxylin-eosin staining. Presence of obscuring inflammation and tangential tissue sectioning are the most common diagnostic pitfalls.

OBJECTIVE: To examine the utility of double immunostaining for cytokeratin-collagen IV or cytokeratin-laminin in the detection of early invasion in VIN 3 and CIN 3. DESIGN: The study group consisted of 10 cases of "VIN 3, suspicious for invasion" and 10 cases of "CIN 3, suspicious for invasion." The negative control group consisted of VIN 3 (n = 15) and CIN 3 (n = 10). The positive control group consisted of cases of invasive vulvar carcinoma (n = 11) and invasive cervical carcinoma (n = 25). All cases were double immunostained for cytokeratin and collagen IV and, in a separate reaction, for cytokeratin and laminin. The continuity of the basement membrane and the presence of stromal invasion were assessed in the stained sections.

RESULTS: The staining for collagen IV and laminin yielded identical results. A well-defined, continuous basement membrane was visualized in all cases of VIN 3 and CIN 3. A discontinuous or absent basement membrane was observed around the malignant cells on the invasive tumor front in all cases of vulvar and cervical carcinoma. In 2 of 10 cases of VIN 3, suspicious for invasion and in 4 of 10 cases of CIN 3, suspicious for invasion definitive foci of microinvasion were identified with the use of double immunostaining. A well-defined, continuous basement membrane was present in the remaining cases "suspicious for invasion."

CONCLUSION: Double immunostaining for cytokeratin- collagen IV or cytokeratin-laminin is useful for evaluation of early invasion in equivocal cases of VIN 3 and CIN 3.
Carcinosarcoma of the Uterine Cervix A Report of Eight Cases With Immunohistochemical Analysis and Evaluation of Human Papillomavirus Status

Am J Surg Pathol 2001;25:338-347

This report describes the morphology, immunohistochemical profiles, and human papillomavirus (HPV) status of eight cervical MMMTs.

Patients' ages ranged from 32 to 93 years (mean, 61 years).

Seven cases showed in situ squamous cell carcinoma (SCC). The invasive epithelial component (EC) was composed of combined adenoid basal carcinoma, basaloid SCC, and adenoid cystic carcinoma (ACC) in two cases. Keratinizing SCC, large cell nonkeratinizing SCC, undifferentiated carcinoma, and basaloid SCC predominated in the remaining tumors, one of which had admixed ACC. The sarcomatous component (SC) was homologous and spindled with admixed myxoid areas in three lesions.

The ECs and SCs in six MMMTs showed dual immunostaining with epithelial membrane antigen and the pan-keratin marker, MNF116. The SC was vimentin-positive in seven cases. Five tumors had a vimentin-positive EC. The SC was positive for muscle specific actin and/or smooth muscle actin in seven lesions, of which four were desmin-positive. Polymerase chain reaction (PCR) using GP5+/GP6+ L1 consensus primers detected HPV DNA in all eight cases. Nonisotopic in situ hybridization with digoxigenin-labeled probes to HPV types 6, 11, 16, 18, 31 and 33 demonstrated integrated HPV 16 in three cases, not only in the EC, but also in nuclei of the SC.

This is the first study to implicate HPV in the evolution of cervical MMMTs. The above observations lend support to a metaplastic theory of histogenesis.

Co-existent Carcinosarcoma and Adenoid Basal Carcinoma of the Uterine Cervix and Correlation with Human Papillomavirus Infection

Yukio Takeshima, M.D.; Vishwa Jeet Amatya, M.B.B.S.; Fumio Nakayori, B.S.; Tomohiro Nakano, C.T.; Yasuhisa Iwaoki, M.D.; Kunihiko Daitoku, M.D.; Kouki Inai, M.D.

From the Second Department of Pathology (Y.T., V.J.A., F.N., T.N., K.I.), Hiroshima University School of Medicine, Japan; and the Departments of Gynecology and Obstetrics (Y.I.), and Internal Medicine (K.D.), Yoshida General Hospital, Japan.

Int J Gynecol Pathol 2002;21:186-190 Abstract quote

A case of carcinosarcoma associated with an adenoid basal carcinoma of the uterine cervix in an 84-year-old woman is described.

The tumor formed a pelvic mass, and total hysterectomy with bilateral salpingo-oophorectomy was performed. The tumor was 17 cm in maximal dimension, arose in the posterior wall of the uterine cervix, and had a solid and cystic, focally myxoid sectioned surface. The tumor was composed of keratinizing squamous cell carcinoma and a mostly nonspecific sarcomatous component with focal chondrosarcomatous differentiation. Multiple foci of adenoid basal carcinoma were observed in the adjacent cervical wall. The HPV L1 gene was detected by PCR in each of the carcinomatous and sarcomatous components of the carcinosarcoma and the adenoid basal carcinoma.

To our knowledge, this is the fifth case of coexisting carcinosarcoma and adenoid basal carcinoma of the uterine cervix in the English language literature.

Detection of Human Papillomavirus DNA and Expression of p16, Rb, and p53 Proteins in Small Cell Carcinomas of the Uterine Cervix.

Wang HL, Lu DW.

Lauren V. Ackerman Laboratory of Surgical Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO.
Am J Surg Pathol. 2004 Jul;28(7):901-8. Abstract quote  

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of primary small cell carcinoma of the uterine cervix, a rare but highly aggressive malignancy. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In squamous cell carcinoma and adenocarcinoma of the cervix, HPV infection is also associated with overexpression of p16, a cyclin-dependent kinase inhibitor.

In this study, 22 cases of primary small cell carcinoma of the uterine cervix were subjected to broad-spectrum HPV DNA amplification and typing, and immunohistochemically examined for the expression of p16, Rb, and p53 proteins.

The results show that HPV DNA was detected in every case (100%), with 18 cases (82%) harboring type 18. The tumor cells exhibited strong nuclear staining for p16 in 20 cases (91%). This was associated with a complete loss of Rb nuclear staining in tumor cells in 16 cases (73%). The p53 protein was essentially undetectable in all cases. In contrast, HPV DNA was not detected in 9 colorectal and 8 urinary bladder small cell carcinomas included in this study for comparison. While similar p16 and Rb expression patterns were observed in these HPV-negative tumors, a different expression pattern for p53 was noted where strong nuclear staining was seen in 8 cases (47%; P = 0.0004 compared with cervical tumors).

These observations indicate that different mechanisms are involved in the pathogenesis of small cell carcinomas of the uterine cervix and support the notion that nuclear p16 overexpression serves as an indication of Rb defunctioning in tumor cells, which may or may not result from high-risk HPV infection.
Overexpression of c-kit protein is an infrequent event in small cell carcinomas of the uterine cervix.

Wang HL, Lu DW.

1Lauren V Ackerman Laboratory of Surgical Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.
Mod Pathol. 2004 Jun;17(6):732-8. Abstract quote  

Primary small cell carcinomas of the uterine cervix are uncommon but highly aggressive malignancies. The recent observations that c-kit proto-oncogene, a tyrosine kinase, is overexpressed in small cell lung cancers and that advanced c-kit-expressing gastrointestinal stromal tumors were successfully treated with a selective tyrosine kinase inhibitor STI-571 (Gleevec, imatinib mesylate) prompted us to investigate c-kit protein expression in cervical small cell carcinomas.

Using a polyclonal antibody against c-kit protein (CD117), our immunohistochemical studies demonstrated a cytoplasmic staining in six of 22 cases (27%) of cervical small cell carcinoma. However, in five of these c-kit-positive cases, the immunoreactivity was focal and/or weak. Only one case (5%) exhibited a strong and diffuse staining pattern comparable to that seen in gastrointestinal stromal tumors. This was in contrast to small cell lung cancers where a positive staining for c-kit was observed in nine of 14 cases (64%) included in the study for comparison. Among them, five (36%) exhibited a strong and diffuse staining pattern. These results indicate that overexpression of c-kit protein is an infrequent event in small cell carcinomas of the uterine cervix.

In comparison with small cell lung cancers, the findings presented in this report may reflect the difference in etiopathogenetic mechanisms underlying these two types of small cell carcinomas.

Small cell carcinoma of the cervix: A clinicopathologic and immunohistochemical study of 23 cases.

Conner MG, Richter H, Moran CA, Hameed A, Albores-Saavedra J.

Departments of Pathology and Obstetrics and Gynecology, The University of Alabama at Birmingham, AL; and the Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX.


Ann Diagn Pathol 2002 Dec;6(6):345-8 Abstract quote

Twenty-three patients with primary small cell carcinoma of the uterine cervix are presented. Their ages ranged between 23 and 63 years (average, 43 years). Blood spotting or vaginal bleeding was the most common clinical presentation. Histologically, the tumors were densely cellular and showed trabecular nesting or a sheet-like pattern. The neoplastic cells had scant cytoplasm, round nuclei, absence of nucleoli, and finely dispersed chromatin. Nuclear molding, single cell necrosis, and high mitotic activity were found in all tumors. There was a minor component of large cell neuroendocrine carcinoma in three cases, while foci of adenocarcinoma were identified in two cases.

Immunohistochemical studies were performed in all 23 tumors which showed immunoreactivity for cytokeratin. Ten small cell carcinomas were immunoreactive for chromogranin, 13 for synaptophysin, and 10 expressed p53 protein. Treatment modalities included hysterectomy alone or combined with chemotherapy and/or radiation therapy. A few patients received chemotherapy and/or radiation alone. Follow-up information was obtained in 22 cases; 15 patients died of tumor between 6 and 43 months, while seven patients have remained alive 12 to 273 months. One patient was lost to follow-up. Small cell carcinoma of the cervix is a highly aggressive neoplasm. However, early diagnosis and combined therapeutic modalities may lead to longer survival in some patients.

Although the use of immunohistochemistry may be helpful in the diagnosis, small cell carcinoma still remains a morphologic diagnosis.



Signal-Amplified Colorimetric In Situ Hybridization for Assessment of Human Papillomavirus Infection in Cervical Lesions

Peter Birner, M.D., Barbara Bachtiary, M.D., Bettina Dreier, Ph.D., Monika Schindl, M.D., Elmar A. Joura, M.D., Gerhard Breitenecker, M.D. and Georg Oberhuber, M.D.

Institute of Clinical PathologyDepartment of Gynecopathology and Cytology (PB, MS, GB, GO); Department of Radiotherapy and Radiobiology (BB, BD); and Department of Gynecology and Obstetrics (EAJ), University of Vienna, Austria

Mod Pathol 2001;14:702-709 Abstract quote

Detection and typing of human papillomavirus (HPV) infection may have a major impact in cervical-screening and follow-up.

In this study various commercially available techniques for the detection of HPV were evaluated. HPV-status was determined in 86 samples of cervical cancer by PCR and direct sequencing, catalyzed signal amplified colorimetric DNA in situ hybridization (CSAC- ISH) (GenPoint system, DAKO), immunohistochemistry (IHC) and in 12 selected cases also by conventional, non-amplified ISH.

Twenty-one samples of cervical intraepithelial neoplasias grade III (CIN III) were investigated by CSAC-ISH, conventional ISH and by IHC, in corresponding PAP smears HPV-detection and typing was performed by CSAC-ISH and Hybrid Capture test II (HC). In additional 20 PAP smears HPV typing was performed using HC and a novel immunocytochemical system for HPV detection and-typing. CSAC-ISH showed good correlation with PCR analysis in cervical cancers: In 87% of PCR positive cases, HPV infection was also detected by CSAC- ISH (66/76). HPV 16 was detected in 75% of PCR-positive cases (44/59), HPV 18 in 71% of PCR positive cases (5/7). CSAC-ISH detected HPV 31 in only 29% of PCR positive cases (2/7), and HPV 33 in 64% of PCR-positive cases (23/36). Nevertheless, CSAC-ISH- false negative cases for HPV 31 or 33 were nearly always combined infections with other HPV types, which were detectable by CSAC-ISH in most cases. CSAC-ISH revealed HPV infection in 20 of 21 HC-positive cervical smears, while in corresponding biopsies (CIN III) CSAC-ISH detected 100% of HPV infections.

Conventional, non-amplified ISH showed significantly lower sensitivity compared with CSAC-ISH, and immunocyto- and -histochemistry were of very low sensitivity for detection of HPV. CSAC-ISH is an easy-to-handle method for detection and typing of cervical HPV infection, and shows sufficient sensitivity for clinical practice.


Biotinyl-Tyramide-Based In Situ Hybridization Signal Patterns Distinguish Human Papillomavirus Type and Grade of Cervical Intraepithelial Neoplasia

Mark F. Evans, Ph.D., Sharon L. Mount, M.D., Barbara G. Beatty, Ph.D. and Kumarasen Cooper, M.B.Ch.B., D.Phil.

Department of Pathology, University of Vermont, Burlington, Vermont


Modern Pathology 2002;15:1339-1347 Abstract quote

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide–based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus.

Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P = .025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P < .0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus–positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P < .0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas.

Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide–based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.


Adv Anat Pathol 2001;8:83-92

Difficulty in separating dysplastic lesions from atrophy or reactive atypia

Ki-67 (MIB-1) and PCNA

LSIL vs. reactive changes
Atrophy vs. HSIL
Immature metaplasia vs. HSIL

Interpretation linked to staining index with a precise cutoff between reactive and lesional epithelium not always possible

MIB-1 staining patterns:
Mature squamous mucosa-nuclei of second and third layers with occasional basal staining
LGSIL-Areas of cytopathic effect in upper epithelial layers
HGSIL-Diffuse staining with higher index of cell nuclei
Atrophy-Markedly reduced or absent staining

Cell cycle and/or proliferation markers: what is the best method to discriminate cervical high-grade lesions?

Lorenzato M, Caudroy S, Bronner C, Evrard G, Simon M, Durlach A, Birembaut P, Clavel C.

Laboratoire Pol Bouin, Department of Histology and Cystology, Maison Blanche Hospital, 51092 Reims Cedex, France.
Hum Pathol. 2005 Oct;36(10):1101-7. Related Articles, Links

The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL.

Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%.

Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium.

Ki-67, Cyclin E, and p16INK4 Are Complimentary Surrogate Biomarkers for Human Papilloma Virus-Related Cervical Neoplasia

Jeffrey T. Keating, M.D.; Aida Cviko, M.D.; Sabine Riethdorf, M.D.; Lutz Riethdorf, M.D.; Bradley J. Quade, M.D., Ph.D.; Deqin Sun, M.S.; Stefan Duensing, M.D.; Ellen E. Sheets, M.D.; Karl Munger, Ph.D.; Christopher P. Crum, M.D.

From the Division of Women's and Perinatal Pathology, Department of Pathology (J.T.K., A.C., B.J.Q., D.S., S.D., E.E.S., K.M., C.P.C.), Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.; and the Department of Gynecopathology (S.R., L.R.), Hamburg, Germany.

Am J Surg Pathol 2001;25:884-891 Abstract quote

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia.

This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data.

Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively.

Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively.

Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

Diagnostic Accuracy of Cervical Low-Grade Squamous Intraepithelial Lesions Is Improved With MIB-1 Immunostaining

Edyta C. Pirog, M.D. ; Rebecca N. Baergen, M.D. ; Robert A. Soslow, M.D. ; Diane Tam, M.D. ; Amy E. DeMattia, M.D. ; Yao-Tseng Chen, M.D. , Ph.D. ; Christina Isacson, M.D.

From the Department of Pathology (E.C.P., R.N.B., D.T., Y.-T.C.), Weill Medical College of Cornell University, the Department of Pathology (R.A.S.), Memorial Sloan-Kettering Cancer Center, and the Department of Pediatrics (A.E.D.), Mt. Sinai School of Medicine, New York, NY; and the Department of Pathology (C.I.), Virginia Mason Medical Center, Seattle, Washington, U.S.A.

Am J Surg Pathol 2002;26:70-75 Abstract quote

There is considerable interobserver variation in the diagnosis of low-grade squamous intraepithelial lesion that involves mature squamous epithelium.

Our aim was to evaluate the utility of MIB-1 immunostaining as an adjunct test to increase diagnostic accuracy. Consecutive cervical biopsies originally diagnosed as normal (n = 26) or low-grade squamous intraepithelial lesion (n = 23) were reviewed by three pathologists to obtain a consensus diagnosis. MIB-1 immunostaining was performed, and positive staining was defined as a cluster of at least two stained nuclei in the upper two thirds of the epithelial thickness. Human papillomavirus (HPV) DNA detection was performed using a polymerase chain reaction assay. All cases were subsequently reclassified as low-grade squamous intraepithelial lesion (LSIL) or normal (NL) when two or three of three gold standard criteria were satisfied (LSIL gold standard criteria = consensus diagnosis of LSIL, HPV+, MIB-1+; NL gold standard criteria = consensus diagnosis of NL, HPV–, MIB-1–).

Using the gold standard diagnoses, we have identified that 14 normal cases (36%) were originally overdiagnosed as LSIL, and one LSIL case (10%) was originally underdiagnosed as normal. All MIB-1-positive cases were HPV+ and identified as LSIL in the consensus review. All MIB-1-negative cases were NL by gold standard criteria. The sensitivity (1.0) and the specificity (1.0) of MIB-1 staining for identifying LSIL were superior to the sensitivity (0.9) and the specificity (0.8) of HPV testing.

In conclusion, MIB-1 is a highly sensitive and specific marker for identifying low-grade squamous intraepithelial lesion and is helpful in verifying the diagnosis of equivocal cases.

Cyclin E
High index of leukocyte cytoplasmic staining requires careful interpretation with significant inflammation

Keratins 8, 10, 13, and 17 are useful markers in the diagnosis of human cervix carcinomas.

Carrilho C, Alberto M, Buane L, David L
Hum Pathol. 2004 May;35(5):546-51. Abstract quote

Several candidate tumor markers for cervical neoplasia have been identified. Among those are keratin markers, whose precise implications in the diagnosis are still under debate.

In the present study, we aimed to clarify the usefulness of studying the expression of keratins 8, 10, 13, and 17 for diagnostic purposes in human cervix carcinomas. Forty-four invasive squamous carcinomas, 10 cervical intraepithelial neoplasia grade III (CIN III), and 10 reference cervix were examined immunohistochemically with monoclonal antibodies. Expression of keratins in reference exocervix, CIN III, and invasive carcinomas was as follows: keratin 8-0, 44.4%, and 57.1%, respectively; keratin 10-77.8%, 40%, and 19%, respectively; keratin 13-100%, 22.2%, and 25%, respectively; keratin 17-0, 40%, and 73.2%, respectively. In invasive carcinomas, expression of keratin 10 was significantly associated with keratinizing carcinomas.

In conclusion, we observed that expression of keratins 8 and 17 and loss of keratins 10 and 13 are good markers of malignant transformation in human cervix. Keratin expression patterns, namely expression of keratin 10, can be useful for subtyping and grading squamous cell carcinomas of the cervix.

Immunohistochemical visualization of histone H1 phosphorylation in squamous intraepithelial lesions of the gynecologic tract.

Kafanas A, Wang BY, Kalir T, Gan L, Bodian C, Fish H, Kohtz DS, Burstein DE.

Departments of Pathology and Biomathematics and the Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY and the Department of Pathology, Serras General Hospital, Serras, Greece.


Hum Pathol 2003 Feb;34(2):166-73 Abstract quote

Immunohistochemical staining was performed on gynecologic tract squamous intraepithelial lesions using a novel phosphorylation-specific monoclonal antibody (designated 12D11) that detects histone H1 when phosphorylated at a cyclin-dependent kinase (CDK)-responsive epitope.

Findings were compared to immunostaining by MIB-1, an extensively studied antibody probe of proliferation. Routinely fixed and processed archival sections were subjected to distinct antigen retrieval and staining protocols for each antibody and were processed for immunodetection of either Ki-67 (with MIB-1) or phosphohistone H1, using a streptavidin-biotin kit and diaminobenzidine as chromagen. For 12D11 staining, antigen retrieval was performed at pH 4.0, and the antibody incubation buffer was supplemented with 1.0 M NaCl. Both 12D11 and MIB-1 stained parabasal cells in normal squamous epithelium.

Staining by 12D11 and MIB-1 of cells in progressively higher strata was found to correlate with the severity of lesions. The mean proportion of positively stained cells was higher in MIB-1-stained sections than in 12D11-stained sections in normal squamous epithelium and in all grades of squamous intraepithelial lesions.

We conclude that the changes in expression patterns of CDK-phosphorylated histone H1 in the spectrum of gynecologic squamous intraepithelial lesions are similar to staining patterns obtained with the proliferation probe MIB-1. The differing proportion of cells stained by MIB-1 and 12D11 suggests that phosphohistone H1 may be a useful alternative proliferation marker that detects a different subpopulation of cycling cells in premalignant squamous lesions.

Increased expression of matriptase is associated with histopathologic grades of cervical neoplasia.

Lee JW, Yong Song S, Choi JJ, Lee SJ, Kim BG, Park CS, Lee JH, Lin CY, Dickson RB, Bae DS.
Hum Pathol. 2005 Jun;36(6):626-33. Abstract quote  

Summary Matriptase is an epithelial-derived, integral serine protease that has been implicated in the progression of epithelial tumors.

We investigated whether the expression of matriptase is associated with the progression of cervical neoplasia. Using immunohistochemistry, we evaluated the matriptase expression in 89 formalin-fixed paraffin-embedded cervical tissues that included 10 normal cervical specimens, 19 low-grade squamous intraepithelial lesions, 20 high-grade squamous intraepithelial lesions, 20 invasive squamous cell carcinomas (ISCC) without lymph node (LN) metastasis, and 20 ISCC with lymph node metastasis.

We also used the reverse transcriptase-polymerase chain reaction technique to determine the expression of matriptase transcripts in normal cervical and ISCC tissues. The immunohistochemical staining showed that the expression of matriptase was undetectable in all normal cervical squamous epithelia, but had cytoplasmic and membranous staining in the normal endocervical glands. Staining gradually increased in accordance with the histopathologic grades from low-grade squamous intraepithelial lesions to high-grade squamous intraepithelial lesions and ISCC ( P < .001); matriptase was detected in most cases (95%) of ISCC. In addition, matriptase transcripts were expressed in all (n = 26) of the ISCC cases by microdissection and reverse transcriptase-polymerase chain reaction, whereas none of the normal squamous epithelia cases (n = 3) expressed matriptase transcripts.

These results suggest that matriptase may play a significant role in the development of cervical carcinoma and may serve as a useful marker of the malignant transformation of cervical squamous cells. Further studies could potentially lead to the development of novel approaches for early detection and therapy for this disease.
Reduced to variable in LGSIL
p16INK4A Immunohistochemistry is Superior to HPV In Situ Hybridization for the Detection of High-risk HPV in Atypical Squamous Metaplasia.

Department of Pathology, Stanford University School of Medicine, Stanford, CA.


Am J Surg Pathol. 2007 Jan;31(1):33-43 Abstract quote

In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16 can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established.

We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16 IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16 IHC.

In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16 IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16 reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16 reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV.

In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16 was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16 reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16 reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means.

Overall, p16 IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.
Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

Benevolo M, Mottolese M, Marandino F, Vocaturo G, Sindico R, Piperno G, Mariani L, Sperduti I, Canalini P, Donnorso RP, Vocaturo A.

1Department of Pathology, Regina Elena Cancer Institute, Rome, Italy.

Mod Pathol. 2006 Mar;19(3):384-91. Abstract quote  

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated.

In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected.

Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.
p16INK4A Immunoexpression and HPV In Situ Hybridization Signal Patterns: Potential Markers of High-Grade Cervical Intraepithelial Neoplasia.

Kalof AN, Evans MF, Simmons-Arnold L, Beatty BG, Cooper K.

From the University of Vermont, Burlington, VT.

Am J Surg Pathol. 2005 May;29(5):674-9. Abstract quote  

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN.

This study examines the correlation between p16 immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16 immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P < 0.001). p16 immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P < 0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration.

Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.
Histologic and Immunohistochemical Characteristics of Neoplastic and Nonneoplastic Subgroups of Atypical Squamous Lesions of the Uterine Cervix

Chisa Aoyama, MD, etal.
Am J Clin Pathol 2005;123:699-706 Abstract quote

Atypical squamous lesion (ASL), a histologic diagnosis of unclear significance in the uterine cervix, can be divided into neoplastic and nonneoplastic groups.

We aimed to determine the morphologic characteristics of these 2 groups. Histologic and immunohistochemical features were evaluated on the original biopsy specimen from 37 ASL cases, and the results were compared between neoplastic (19 cases) and nonneoplastic (18 cases) groups, which were determined based on the follow-up histopathologic findings.

Mitosis, vertical nuclear growth pattern, no perinuclear halo, indistinct cytoplasmic border, primitive cells in the upper third of the squamous layer, p16+ cells in the upper two thirds of the squamous layer, and Ki-67+ cells in the upper two thirds of the squamous layer were significant indicators for neoplastic ASLs. Of the 19 neoplastic ASLs, 16 (84%) had 5 or more of these 7 indicators. The majority (16/18 [89%]) of the nonneoplastic ASLs had 2 or fewer indicators.

Determination of the histologic and immunohistochemical characteristics is useful for distinguishing neoplastic and nonneoplastic ASLs.
p16INK4A and p14ARF expression pattern by immunohistochemistry in human papillomavirus-related cervical neoplasia.

Wang JL, Zheng BY, Li XD, Nokelainen K, Angstrom T, Lindstrom MS, Wallin KL.

[1] 1Department of Molecular Medicine, Karolinska Institute, Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden [2] 5Department of Gynecology, Peking University People's Hospital, Beijing, China.

Mod Pathol. 2005;18:629-637 Abstract quote

Human papillomavirus is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer.

The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of human papillomavirus DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4A and possibly p14ARF have been proposed as putative surrogate biomarkers that would allow identification of dysplastic cervical epithelia. Serial consecutive biopsies representing normal cervical epithelium to cervical intraepithelial neoplasia and/or invasive cervical cancer were stained with immunohistochemistry for p16INK4A, p14ARF and proliferating cell nuclear antigen. The positive rates of these markers were significantly higher in cervical intraepithelial neoplasia and in squamous cell carcinoma than in normal cervix (P<0.01). No significant difference was noted between lesions progressing from cervical intraepithelial neoplasia to squamous cell carcinoma for both p16INK4A and p14ARF expression (P>0.05).

For both biomarkers, nuclear staining was predominantly seen. However, the cytoplasmic stain of p16INK4A increased with disease progression and the pattern of expression varied between different tumors and its location within the lesion. Both nuclear and cytoplasmic staining with p16INK4A and p14ARF of affected epithelial cells were considered positive. In the adjacent normal tissue to cervical neoplasia, the positive rates of p16INK4A, p14ARF and proliferating cell nuclear antigen expression were higher than those found distant to these lesions but the findings did not reach statistical significance.

No correlation was seen between the human papillomavirus types detected and the expression of p16INK4a and p14ARF. In conclusion, overexpression of p16INK4A and p14ARF act as potential biomarkers for cervical cancer progression from premalignant lesions.
Human papillomavirus genotyping and p16(INK4a) expression in cervical intraepithelial neoplasia of adolescents.

Hu L, Guo M, He Z, Thornton J, McDaniel LS, Hughson MD.

1Department of Pathology, The University of Mississippi Medical Center, Jackson, MS, USA.

Mod Pathol. 2005;18:267-273 Abstract quote

Adolescents have high rates of human papillomavirus (HPV) infection, and persistent high-risk HPV infection can lead to the development of cervical cancer. The cyclin-dependent kinase inhibitor, p16(INK4a) is overexpressed in cervical intraepithelial neoplasia (CIN), probably due to a persistent and integrated HPV infection.

This study investigated p16(INK4a) expression, grades of CIN, and high-risk HPV infection in adolescent cervical biopsies. Biopsies were immunohistochemically stained for p16(INK4a). The presence of wide-spectrum, low-risk, or high-risk HPV was determined by amplifying DNA extracted from the cervical biopsies. Biopsies were classified as cervicitis, 15 cases; CIN 1, 48 cases; CIN 2, 46 cases, and CIN 3, 52 cases. The distribution of p16(INK4a) staining was graded as patchy, diffuse basal, and diffuse full thickness. Pearson's chi(2) tests analyzed the relationships between p16(INK4a) staining, HPV infection, and CIN. Biopsies of cervicitis were negative for HPV and for p16(INK4a) expression. High-risk HPV 16, 18, and 31 increased from 18% in CIN 1 to 66% in CIN 2/3 (P<0.001). In CIN 1, p16(INK4a) was positive in 44% of biopsies with 35% showing patchy, 7% diffuse basal, and one case (2%) showing diffuse full thickness staining. In CIN 2/3, p16(INK4a) was positive in 97% of biopsies with 23% showing patchy, 21% diffuse basal, and 53% diffuse full thickness staining.

The difference in the proportions of biopsies showing patchy p16(INK4a) staining in CIN 1 and diffuse full thickness staining in CIN 2/3 was significant (P<0.001). In CIN 1, 61% of high-risk HPV-positive biopsies were p16(INK4a) negative, while all high-risk HPV-positive CIN 2/3 biopsies were p16(INK4a) positive. Diffuse, full thickness p16(INK4a) expression discriminated low-grade from high-grade CIN and appears to be a marker of persistent high-risk HPV infection
The Predictive Value of p16INK4a and Hybrid Capture 2 Human Papillomavirus Testing for High-Grade Cervical Intraepithelial Neoplasia

Ming Guo, MD, Lulin Hu, MD, Mithra Baliga, MD, Zhi He, MD, and Michael D. Hughson, MD
Am J Clin Pathol 2004;122:894-901 Abstract quote

We performed p16INK4a immunocytochemical analysis and Hybrid Capture 2 (HC2, Digene, Gaithersburg, MD) high-risk HPV testing on 210 abnormal SurePath (TriPath Imaging, Burlington, NC) Papanicolaou specimens diagnosed as low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL).

The results were compared with 121 follow-up biopsy specimens. p16INK4a was positive in 57.9% of women with LSIL compared with 97.1% of women with HSIL. In contrast, HC2 testing was positive in 85.0% of women with LSIL and 86.4% of women with HSIL. The differences in the positive rates for16INK4a between LSIL and HSIL was significant (P < .001), whereas for HC2 it was not (P = .264). In patients who had cervical biopsies following a cytologic diagnosis of LSIL, the positive predictive value (PPV) of p16INK4a for a biopsy of cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3; 33%) was significantly higher than the PPV of HC2 results (21%) (P < .001).

Using liquid-based cytology specimens, p16INK4a immunocytochemical analysis has a higher PPV than reflex HC2 HPV testing for identifying CIN2/3 among patients with LSIL and might be useful for selecting patients with LSIL for colposcopy.
Evaluation of p16 and pRb expression in cervical squamous and glandular neoplasia.

Tringler B, Gup CJ, Singh M, Groshong S, Shroyer AL, Heinz DE, Shroyer KR
Hum Pathol. 2004 Jun;35(6):689-96. Abstract quote  

p16(INK4a) is known to play a critical role as a negative regulator of cell cycle progression and differentiation by controlling the activity of the tumor-suppressor protein pRb. The present study evaluated the expression of p16(INK4a) and pRb in cervical squamous and glandular neoplasia.

Immunohistochemical staining was performed for p16(INK4a) and pRb in formalin-fixed, paraffin-embedded tissue sections of the uterine cervix using an indirect immunoperoxidase method. p16(INK4a) staining was detected in 7 of 108 sections (6.5%) of normal squamous mucosa, in scattered ciliated columnar cells in 33 of 88 sections (37.5%) of normal endocervical glands, in 9 of 30 sections (30%) with Nabothian cysts, and in 4 of 4 areas (100%) of tubal metaplasia.

In contrast, strong p16(INK4a) staining was found in 13 of 18 cases (72.2%) of cervical intraepithelial neoplasia (CIN) I and in all cases of CIN II/III (n = 46), squamous cell carcinoma (n = 18), endocervical glandular dysplasia (n = 10), adenocarcinoma in situ (n = 23), and invasive adenocarcinoma (n = 12). pRb expression was detected in each diagnostic category; however, the proportion of pRb-positive cells was relatively decreased in high-grade premalignant and malignant lesions of the squamous and endocervical mucosa and showed a generally inverse correlation with the expression of p16(INK4a) at the tissue level.

These findings confirm a correlation between the expression of p16(INK4a) and pRb in cervical neoplasias and indicate that p16(INK4a) is a specific marker for premalignant and malignant lesions of the squamous and endocervical mucosa.
Combined p53 and Retinoblastoma Protein Detection Identifies Persistent and Regressive Cervical High-Grade Squamous Intraepithelial Lesions.

Baak JP, Kruse AJ, Garland SM, Skaland I, Janssen EA, Tabrizi S, Fagerheim S, Robboy S, Nilsen ST.

From the Departments of *Pathology and daggerGynecology, Stavanger University Hospital, Stavanger, Gade Institute; double daggerUniversity of Bergen, Bergen, Norway; section signFree University, Amsterdam, The Netherlands; parallelDepartment of Microbiology & Infectious Diseases, Royal Women's Hospital, Melbourne, Australia; paragraph signDepartment of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia; and #Department of Pathology, Duke University Medical Center, Durham, NC.
Am J Surg Pathol. 2005 Aug;29(8):1062-1066. Abstract quote  

Most cervical high-grade squamous intraepithelial lesions (HSILs) persist, but approximately one third regress (ie, no HSIL in follow-up biopsies).

To identify factors related to histologic proven persistence or regression. Twenty-eight small histologic (marker) biopsies with adequate follow-up were analyzed for human papillomavirus (HPV) genotypes and different immunoquantitative proliferation, cell cycle regulation, and differentiation markers. All cases had a biopsy-interval between the marker and first follow-up biopsy of at least 100 days (median, 8.2 months; range, 3.4-22.5 months). Follow-up was classified as regression or persistence. All lesions were high-risk (hr) HPV and p16 positive, 63% for HPV-16 or HPV-16 mixed with other hr genotypes, while 37% had other hrHPV types.

The marker biopsies of the persistent HSILs had lower p53 and retinoblastoma protein (pRb) detected in the deep half of the epithelium (P = 0.001 and 0.02, respectively) than nonpersistent HSILs. The degree of positivity of p16, Ki-67, cyclin D1, lesion extent, positivity of the resection margins, and patient age were all unrelated to persistence or regression. Lesions with HPV-16 or mixed-16 genotypes had a significantly lower percentage of pRb (P = 0.02), p53 (P = 0.02), and cyclin D (P = 0.04) positive nuclei in the deep epithelial layers.

In agreement with this, type-16 positive HSILs had a lower regression percentage than those with other HPV types, but the difference was not significant. HSILs with combined negativity/low positivity for p53 and pRb protein in small histologic biopsies are highly likely to persist, contrasting those in which one of these cell cycle regulators is strongly positive (p53 > 15%; pRb > 40%).

Telomerase and human papillomavirus as diagnostic adjuncts for cervical dysplasia and carcinoma.

Jarboe EA, Thompson LC, Heinz D, McGregor JA, Shroyer KR.

Department of Pathology, University of Colorado Health Sciences Center, Denver, CO, USA.
Hum Pathol. 2004 Apr;35(4):396-402. Abstract quote  

Telomerase and human papillomavirus (HPV) DNA were evaluated as potential markers of high-grade dysplasia in cervical cytological specimens. Cytology specimens were collected from patients at the time of colposcopic evaluation for management of a previous abnormal cytology test result.

Telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP), and HPV DNA was detected by polymerase chain reaction with L1 consensus-sequence primers and filter hybridization genotyping. Telomerase was detected in 8 of 97 (8.2%) cases with normal cytology or benign cellular changes, in 7 of 98 (7.1%) cases of atypical squamous cells of undetermined significance (ASCUS), in 3 of 95 (3.2%) cases of low-grade squamous intraepithelial lesion (LSIL), and in 17 of 48 (35.4%) cases with high-grade squamous intraepithelial lesion (HSIL). High-risk HPVs were detected in 23 of 97 (23.7%) cases with normal/reactive cellular changes (RCC) cytology, in 28 of 98 (28.6%) cases of ASCUS, in 69 of 95 (72.6%) cases of LSIL, and in 35 of 48 (72.9%) cases of HSIL. Telomerase expression did not correlate with the detection of high-risk HPVs in any cytological diagnostic categories.

Telomerase and HPV test results of cytological specimens were correlated with the histological diagnoses of concurrent cervical biopsy specimens. Telomerase showed a sensitivity of 29.9% and a specificity of 94.0% for biopsy-confirmed cervical intraepithelial neoplasia (CIN) II/III. In contrast, high-risk HPVs were detected in 70.1% of cases with underlying CIN II/III, with a specificity of 62.5%. A relatively high proportion of normal/RCC or ASCUS cases with telomerase-positive test results had underlying high-grade dysplasia on cervical biopsy.

Thus, technical and practical limitations of the TRAP assay in cervical cytology specimens limit the practical application of telomerase as a diagnostic adjunct in cervical cytopathology.

p16INK4a Immunohistochemistry Improves Interobserver Agreement in the Diagnosis of Cervical Intraepithelial Neoplasia.

Klaes R, Benner A, Friedrich T, Ridder R, Herrington S, Jenkins D, Kurman RJ, Schmidt D, Stoler M, Von Knebel Doeberitz M.


Am J Surg Pathol 2002 Nov;26(11):1389-99 Abstract quote

It has been repeatedly shown that there is a substantial lack of interobserver reproducibility in the histologic diagnosis of cervical intraepithelial neoplasia (CIN), which might be improved by a more specific diagnostic biomarker. Cervical cancer and CIN, but not other cervical epithelia, express high levels of the cyclin-dependent kinase inhibitor p16, suggesting that staining for this marker could help to more precisely identify CIN in tissue sections and therefore reduce variation in interpretation of cervical lesions.

To test this hypothesis, 194 cervical cone biopsy samples were selected from a routine histopathology laboratory. Two consecutive sections from each biopsy were stained with hematoxylin and eosin and with a p16 -specific monoclonal antibody, respectively. Five experienced cervical pathologists examined the slides. The agreement in the diagnosis between pairs or groups of observers was calculated by kappa statistics. Significant discrepancies were observed in the diagnostic interpretation of hematoxylin and eosin-stained slides, particularly for low-grade lesions (kappa value 0.60 [95% confidence interval 0.58-0.63]). There was significantly better agreement in the interpretation of p16 expression (kappa value 0.91 [95% confidence interval 0.84-0.99]). Expression of p16 was restricted to CIN 2/CIN 3, CIN 1 associated with high-risk human papillomavirus, or cervical cancer. p16 immunostaining allowed precise identification of even small CIN or cervical cancer lesions in biopsy sections and helped to reduce interobserver variation in the histopathologic interpretation of cervical biopsy specimens.

Thus, p16 immunohistochemistry can reduce false-negative and false-positive biopsy interpretation and thereby significantly improve cervical (pre)-cancer diagnosis.

Evaluation of MIB-1-Positive Cell Clusters as a Diagnostic Marker for Cervical Intraepithelial Neoplasia

Arnold-Jan Kruse, M.D.; Jan P. A. Baak, M.D., Ph.D., F.R.C.Path., F.I.A.C. (Hon), Dr. HC (Antwerp); Tove Helliesen, M.D.; Kjell H. Kjellevold, M.D.; Marco G. W. Bol, M.D.; Emiel A. M. Janssen, M.Sc.

Am J Surg Pathol 2002; 26(11):1501-1507 Abstract quote

The objects of the study were to evaluate MIB-1-positive cell clusters (MIB-C) for distinguishing normal, reactive, and cervical intraepithelial neoplasia (CIN) biopsies and to determine possible pitfalls.

Seventy-seven consecutive cervical specimens routinely diagnosed (Dx_orig) as CIN 1 or 2, or no-CIN, were revised independently by two expert gynecopathologists. MIB-1 staining and oncogenic human papillomavirus (HPV) assessment (by polymerase chain reaction) were performed. Independent diagnoses (plus oncogenic HPV status, in case of disagreement between the experts) were used to obtain a final diagnosis (Dx_final) and compared with MIB-C. Four of the 27 (15%) Dx_final = normal were HPV positive. Agreement between the gynecopathologists was 72 of 77 (94%). There were 30 (39%) discrepancies between Dx_orig and Dx_final (23 = 30% downgrades and 7 = 9% upgrades). All 23 downgrades were HPV negative and all seven upgrades were HPV positive.

Overall agreement between Dx_orig and MIB-C was 73%, and with Dx_final 99%. Sensitivity, specificity, and positive and negative predictive values of MIB-C were very high without false negatives. Tangential cutting of MIB-1-positive parabasal cells and inflammatory cells can erroneously be overdiagnosed as a MIB-C. One single false positive of the 48 non-CIN cases (an immature squamous metaplasia) showed a special, easily recognizable MIB-1 pattern, different from CIN because the MIB-1 staining in the nuclei is not diffuse (as in CIN) but clumped. Moreover, positive nuclei are somewhat less densely packed than in CIN.

When tangentially cut parabasal cells and inflammatory cells are carefully excluded, MIB-C is a strong diagnostic adjunct in distinguishing CIN from normal or benign cervical squamoepithelial lesions.



Pagetoid Dyskeratosis of the Cervix An Incidental Histologic Finding in Uterine Prolapse

J. Fernando Val-Bernal, M.D., Ph.D.; Jesús Pinto, M.D.; M. Francisca Garijo, M.D., Ph.D.; M. Soledad Gómez, Pharm.D.

From the Department of Anatomical Pathology, Marqués de Valdecilla University Hospital, Medical Faculty, University of Cantabria, Santander, Spain.

Am J Surg Pathol 2000;24:1518-1523 Abstract quote

Pagetoid dyskeratosis, is considered a reactive process in which a small part of the normal population of keratinocytes is induced to proliferate. The lesion is characterized by pale cells resembling those of Paget's disease within the epidermis. These cells have been seen as an incidental finding in a variety of benign papules most commonly located in intertriginous areas. Among the inductors of the lesion, friction is suspected. To the best of our knowledge, these pale cells have not been reported in the cervix.

We describe the location of the lesion in the ectocervix and the incidence of this lesion in a group of 100 unselected patients surgically treated for uterine prolapse. Another group of 100 unselected patients treated for uterine leiomyoma was used as a control. Pagetoid dyskeratosis was found in 37 cases (37%) of uterine prolapse and in five cases (5%) of uterine leiomyomas. The lesion is more common in uterine prolapse (p <0.001) and is not significantly associated with cornification of the epithelium (p = 0.72343). The cells of pagetoid dyskeratosis show an immunohistochemical profile different from the surrounding squamous cells characterized by premature keratinization. Pagetoid dyskeratosis cells have shown positivity for high molecular weight cytokeratin and negative reaction for low molecular weight cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, and human papilloma virus. Pagetoid dyskeratosis cells must be distinguished from artefactual clear cells, glycogen-rich cells, koilocytes, extramammary Paget's disease cells, and pagetoid spread of carcinoma cells to the cervix. In cases in which pagetoid dyskeratosis shows a florid expression, there is a hazard of overdiagnosis.

The pathologist should be aware of the histologic features of pagetoid dyskeratosis in the ectocervix to avoid misdiagnosis and unnecessary treatment. Routine histologic study is usually sufficient to identify the lesion.


Stratified Mucin-Producing Intraepithelial Lesions of the Cervix Adenosquamous or Columnar Cell Neoplasia?

Jeong-Ja Park, M.D.; Deqin Sun, M.S.; Bradley J. Quade, M.D., Ph.D.; Cythia Flynn, M.D.; Ellen E. Sheets, M.D.; Annie Yang, B.S.; Frank McKeon, Ph.D.; Christopher P. Crum, M.D.

From the Division of Women's and Perinatal Pathology, Department of Pathology, Brigham and Women's Hospital (J.-J.P., D.S., B.J.Q., C.P.C.), Department of Pathology, Massachusetts General Hospital (C.F.), Department of Obstetrics and Gynecology (E.E.S.), Brigham and Women's Hospital, and the Department of Cell Biology (A.Y., F.M.), Harvard Medical School, Boston, Massachusetts, U.S.A.

Am J Surg Pathol 2000;24:1414-1419 Abstract quote

BACKGROUND: Squamous (CIN) and glandular (ACIS) intraepithelial lesions often coexist in the same cervical specimen. However, a less common and little studied variant consists of a stratified epithelium resembling CIN in which conspicuous mucin production is present (Stratified Mucin-producing Intraepithelial LEsions (SMILE). This report describes the phenotypic characteristics of the SMILE, its associated lesions, and its immunophenotype.

METHODS: Eighteen SMILEs were identified by the presence of conspicuous cytoplasmic clearing or vacuoles in lesions otherwise resembling CIN. The morphologic spectrum of SMILEs was detailed; including associated intraepithelial and invasive cervical neoplasms. In addition, selected cases were stained for muci-carmine, markers of squamous cell/reserve cell differentiation (keratin-14 and p63), and proliferative activity (Mib-1).

RESULTS: Stratified neoplastic epithelial cells with a high Mib-1 index and a rounded or lobular contour at the epithelial-stromal interface characterized SMILEs. In contrast to CIN, in which mucin droplets are confined to surface cells, mucin was present throughout the epithelium, varying from indistinct cytoplasmic clearing to discrete vacuoles. SMILEs were distinguished from benign metaplasia by nuclear hyperchromasia and a high Mib-1 index. All but three coexisted with either a squamous (CIN) or glandular (ACIS) precursor lesion. Nine of nine coexisting invasive carcinomas contained glandular, adenosquamous differentiation, or both. SMILEs stained negative for keratin-14 and variably for p63. When present, staining with p63 was confined to basal areas of SMILEs and was absent in areas of columnar differentiation.

CONCLUSIONS: SMILEs are unusual cervical intraepithelial lesions best classified as variants of endocervical columnar cell neoplasia based on immunophenotype. The distribution and immunophenotype of SMILEs are consistent with a neoplasm arising in reserve cells in the transformation zone. The coexistence of a wide spectrum of intraepithelial and invasive cell phenotypes suggests that SMILEs are a marker for phenotypic instability, emphasizing the importance of identifying SMILEs and ensuring a complete examination of specimens containing this unusual precursor lesion.


The Small Blue Cell Dilemma Associated With Tamoxifen Therapy

Yi Jun Yang, MD, PhD, Linda K. Trapkin, DO, Roberta K. Demoski, CT(ASCP), Jeannette Bellerdine, MS, SCT(ASCP), and Celeste N. Powers, MD, PhD

From the Department of Pathology, Upstate University, State University of New York, Syracuse (Dr Yang and Ms Demoski); the Department of Pathology, Laboratory Alliance of Central New York at St Joseph's Hospital, Syracuse (Dr Trapkin and Ms Bellerdine); and the Department of Pathology, Medical College of Virginia, Virginia Commonwealth University, Richmond (Dr Powers)

Arch Pathol Lab Med 2001;125:1047–1050. Abstract quote

Context. —Several endometrial diseases, such as endometrial hyperplasia, endometrial carcinoma, and endometrial polyps, have been reported to be associated with tamoxifen administration. We recently observed a high incidence of distinctive small blue cells in Papanicolaou tests of women who had received tamoxifen treatment for breast carcinoma.

Objectives. —To define the characteristics of these small blue cells, to identify the patient population in which they are found, and to determine the clinical significance and possible etiology of these findings.

Design.—A total of 154 Papanicolaou tests from 60 patients with a clinical history of tamoxifen therapy were reviewed retrospectively.

Results. —Small blue cells were found in 40% of Papanicolaou tests from patients who received tamoxifen therapy. Patients with small blue cells in their Papanicolaou tests were an average of 9 years older at the time tamoxifen therapy was initiated than those without. Among the available follow-up surgical biopsies, no malignant diagnoses were made.

Conclusions. —We conclude that these distinctive small blue cells are found more frequently in older patients and most probably represent proliferative reserve cells of cervical/vaginal epithelium resulting from the estrogenic agonist effect of tamoxifen. More importantly, they are nonneoplastic in nature.



Human papillomavirus infection is transient in young women: a population-based cohort study.

Evander M, Edlund K, Gustafsson A, Jonsson M, Karlsson R, Rylander E, Wadell G.

Department of Virology, Umea University, Sweden.

J Infect Dis 1995 Apr;171(4):1026-30 Abstract quote

The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from a cohort of 276 young women was determined by a general two-step polymerase chain reaction.

HPV infection fluctuated among young women during a 2-year interval. The total prevalence of HPV infection decreased from 21% to 8.3%. The most prevalent HPV types at enrollment were HPV-16 (3.3%) and HPV-6 (2.9%). At follow-up, the most common type was HPV-16 (2.9%), while no HPV-6 was detected. In 2 women only, the same HPV type persisted.

Regression of HPV infection was found in 80% of the women. A new HPV type-specific infection was detected in 7.2% of the women and was independently associated with a new sex partner or an abnormal smear since enrollment.

Transience of cervical HPV infection in sexually active, young women with normal cervicovaginal cytology.

Hinchliffe SA, van Velzen D, Korporaal H, Kok PL, Boon ME.

University Department of Pathology, Royal Victoria Infirmary, Newcastle-upon-Tyne, UK.

Br J Cancer 1995 Oct;72(4):943-5 Abstract quote

Human papillomavirus DNA was detected in cervical specimens from 366 sexually active young women with cytomorphologically normal cervices using the polymerase chain reaction.

In 93% (25/27) of initially infected women, the same viral type was not detected upon re-examination four menstrual cycles later.

These results suggest that the majority of HPV infections in young women are transient.

Natural history of cervicovaginal papillomavirus infection in young women.

Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD.

Department of Epidemiology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

N Engl J Med 1998 Feb 12;338(7):423-8 Abstract quote

BACKGROUND: Genital human papillomavirus (HPV) infection is highly prevalent in sexually active young women. However, precise risk factors for HPV infection and its incidence and duration are not well known.

METHODS: We followed 608 college women at six-month intervals for three years. At each visit, we collected information about lifestyle and sexual behavior and obtained cervicovaginal-lavage samples for the detection of HPV DNA by polymerase chain reaction and Southern blot hybridization. Pap smears were obtained annually.

RESULTS: The cumulative 36-month incidence of HPV infection was 43 percent (95 percent confidence interval, 36 to 49 percent). An increased risk of HPV infection was significantly associated with younger age, Hispanic ethnicity, black race, an increased number of vaginal-sex partners, high frequencies of vaginal sex and alcohol consumption, anal sex, and certain characteristics of partners (regular partners having an increased number of lifetime partners and not being in school). The median duration of new infections was 8 months (95 percent confidence interval, 7 to 10 months). The persistence of HPV for > or =6 months was related to older age, types of HPV associated with cervical cancer, and infection with multiple types of HPV but not with smoking. The risk of an abnormal Pap smear increased with persistent HPV infection, particularly with high-risk types (relative risk, 37.2; 95 percent confidence interval, 14.6 to 94.8).

CONCLUSIONS: The incidence of HPV infection in sexually active young college women is high. The short duration of most HPV infections in these women suggests that the associated cervical dysplasia should be managed conservatively.

Epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer.

Franco EL, Villa LL, Sobrinho JP, Prado JM, Rousseau MC, Desy M, Rohan TE.

Department of Oncology, McGill University, Montreal, QC, Canada H2W 1S6.

J Infect Dis 1999 Nov;180(5):1415-23 Abstract quote

Acquisition and clearance of cervical human papillomavirus (HPV) infection were analyzed among 1425 low-income women attending a maternal and child health program in Sao Paulo, Brazil. Specimens collected every 4 months were tested by a polymerase chain reaction protocol (MY09/11).

In all, 357 subjects were positive at least once. There were 1.3% new infections per month, with 38% cumulative positivity after 18 months. Of 177 positive subjects at enrollment, only 35% remained infected after 12 months. The monthly clearance rate was higher for nononcogenic types (12.2%; 95% confidence interval [CI], 9.6-15.4) than for oncogenic HPV infections (9.5%; 95% CI, 7.5-11.9). Median retention times were 8.1 months (95% CI, 7.8-8.3) for oncogenic types and 4.8 months (95% CI, 3.9-5.6) for nononcogenic HPV infections. The mean infection durations were 8.2 and 13.5 months for nononcogenic and oncogenic types, respectively.

Although a woman's age did not affect mean duration for oncogenic types (13-14 months), nononcogenic-type infections lasted longer (10. 2 months) among younger (<35 years old) than in older women (5.6 months).

Incidence, prevalence, and clearance of type-specific human papillomavirus infections: The Young Women's Health Study.

Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, Papenfuss MR, Abrahamsen M, Inserra P, Olvera S, Hatch K.

Arizona Cancer Center, Tucson 85724-5024, USA.

J Infect Dis 2002 Aug 15;186(4):462-9 Abstract quote

The natural history of type-specific human papillomavirus (HPV) infections was examined in a cohort of 331 women aged 18-35 years who self-referred for routine gynecological care.

Participants underwent a gynecological examination at baseline and at approximately 4 and approximately 10 months after baseline. Cervical samples were collected for HPV testing and genotyping at each visit, as was information on reproductive, sexual, and medical histories. The rate of new HPV infections was 2.9% per month; the highest rates were observed for HPV types 16, 39, 84, and 51. Among women who tested negative for HPV at baseline, the cumulative probability of acquiring an oncogenic HPV strain during a 12-month follow-up period was 0.32, compared with 0.18 for nononcogenic strains.

Women who had had >/=1 new male sex partner in the recent past were significantly more likely to acquire a new HPV infection (relative hazard, 2.39; 95% confidence interval, 1.20-4.76).

The median time to clearance of infection was significantly longer for oncogenic strains (9.8 months) than for nononcogenic strains (4.3 months).


Hum Pathol 2000;31:1357-1362
Although serum levels cannot be used to screen for cervical cancer, immunoperoxidase studies found increased CEA expression in CIN grade III and cervical squamous carcinoma

Suggests that it may be important to identify those at risk for progressive cervical neoplasia


Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix

Shirley A. Southern, PhD
Ian W. McDicken, MD, MRCPath
C. Simon Herrington, DPhil, MRCP, MRCPath

Hum Pathol 2002;32:1351-1355. Abstract quote

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells.

In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle–associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001).

These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.

Expression of Fas Ligand and bcl-2 in Cervical Carcinoma and Their Prognostic Significance

Satoru Munakata, MD, PhD, etal.
Am J Clin Pathol 2005;123:879-885 Abstract quote

Apoptosis-related factors are known to influence survival with many malignant tumors.

We performed immunohistochemical analysis of Fas ligand (FasL) and bcl-2 in invasive cervical cancer to determine the association with prognosis. In 125 patients with cervical cancer, 93 cases (74.4%) were positive for FasL, and 94 cases (75.2%) were positive for bcl-2. When 101 cases, clinical stages I through IV, were analyzed by univariate analysis, negative bcl-2 (P = .035) and combined positive FasL and negative bcl-2 (PFNB; P = .0025) were associated with significantly decreased disease-free survival. Positive FasL (P = .042), negative bcl-2 (P = .0004), and PFNB (P < .0001) were associated with a significantly worse prognosis in overall survival.

In clinical stages II through IV, positive FasL (P = .04), negative bcl-2 (P = .002), and PFNB (P < .0001) had significant associations with shorter disease-free survival and positive FasL (P = .049), negative bcl-2 (P < .0001), and PFNB (P < .0001) with worse overall survival.

MIB-1 labeling index as a prognostic indicator for survival in patients with FIGO stage IB squamous cell carcinoma of the cervix.

Ho DM, Hsu CY, Chiang H.

Department of Pathology and Laboratory Medicine, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China.

Gynecol Oncol 2000 Jan;76(1):97-102 Abstract quote

OBJECTIVE: The goal of this study was to assess whether the MIB-1 labeling index (LI) and conventional parameters could distinguish between a good and a poor prognosis in patients with squamous cell cervical carcinoma of the same FIGO stage.

METHODS: The study included 97 cases of stage IB squamous cell cervical carcinoma which were treated with radical hysterectomy between 1989 and 1991. The relation of the MIB-1 LI, conventional clinicopathologic parameters, and survival was evaluated.

RESULTS: The MIB-1 LI of the entire group of tumors was 57.1 +/- 15.9 (mean +/- SD). The MIB-1 LI was significantly different only in living and dead patients, while it was not related to conventional clinicopathologic prognostic parameters. The 5- and 10-year survival rates of patients with an MIB-1 LI 55, which were 73.1 and 66%, respectively. Multivariate analyses showed that MIB-1 LI >55, lymph node metastasis, and tumor size >/=40 mm were powerful predictors of shorter survival.

CONCLUSIONS: Lymph node metastasis, tumor size, and MIB-1 LI were significant prognostic markers in patients with stage IB squamous cell cervical carcinoma.


Neoadjuvant Chemotherapy in Cervical Carcinoma: Regulators of Cell Cycle, Apoptosis, and Proliferation as Determinants of Response to Therapy and Disease Outcome

Silvano Costa, MD
Patrizia Terzano, MD
Donatella Santini, MD, etal


Am J Clin Pathol 2001;116:729-737 Abstract quote

To evaluate whether cellular markers predict the responsiveness to neoadjuvant chemotherapy (NAC) in cervical cancer, 21 patients with stage I and II cervical carcinomas treated by NAC before surgery were followed up for a mean of 52.3 months.

Pre-NAC biopsy and operative specimens were subjected to counting of apoptotic (AI/V) and mitotic (MI/V) indices, detection of human papillomavirus (HPV) DNA, and immunohistochemical analysis of cell cycle and proliferation markers (p21, p53, pRb, proliferating cell nuclear antigen [PCNA], Ki-67) and multidrug resistance gene (MDR1), as related to NAC response (RAC), recurrence-free (RFS), and overall (OS) survival. Adenosquamous histology and lymph node involvement were significant determinants of nonsurvival. All carcinomas contained HPV DNA. In univariate analysis, p21, pRb, and MDR1 in the biopsy specimen and PCNA, Ki-67, and pRb in the surgical sample significantly predicted RAC, while age, AI/V, number of lymph nodes removed, and MI/V predicted RFS. Highly significant predictors of OS were AI/V, number of lymph nodes removed, post-NAC MDR1 expression, MI/V, and recurrence.

Multivariate analysis confirmed the strong post-NAC effects of histologic type, AI/V, and MDR1 expression for RFS, and recurrence, age, and Ki-67 expression for OS. NAC responders with slightly decreased AI/V and increased MI/V had a poor prognosis.

INTRAOPERATIVE FROZEN SECTIONS ON CERVIX In general, this should not be performed. There are definite limitations with a frozen section preparation not the least of which is frozen section artifact and complete and adequate sampling. Ideally, the surgeon should have the patience to wait until the permanent sections of the cone biopsy can be obtained and the pathologist has a chance to carefully examine the cervix to rule out microinvasion.
Efficacy of Cone Biopsy of the Uterine Cervix During Frozen Section for the Evaluation of Cervical Intraepithelial Neoplasia Grade 3

Mai Gu, MD, PhD, and Fritz Lin, MD
Am J Clin Pathol 2004;122:383-388 Abstract quote

We retrospectively selected 22 cases in which patients with a biopsy-proven diagnosis of cervical intraepithelial neoplasia grade 3 underwent cervical conization for frozen section (FS) evaluation followed by hysterectomy at the University of California Irvine Medical Center, Orange, during the August 1995 to September 9, 2001.

All slides from FS and permanent section (PS) and hysterectomy specimens were reviewed. FS diagnoses were compared with those of previous biopsies, PS, and hysterectomy specimens. The PS correlated with FS in all cases but 1. Appropriate surgery was performed for all patients based on FS diagnosis. The McNemar test was used to compare the results of FS and PS, with a 2-sided P value of 1.0 and a k coefficient of 0.7755 with a 95% confidence level, indicating that the 2 groups were not significantly different.

FS evaluation of cervical conization is as efficacious and accurate as evaluation of regular specimens in providing information for the appropriateness of same-day surgery. We recommend that entire tissue be submitted for FS to avoid sampling errors and to increase diagnostic accuracy.

Reliability of the frozen section in sharp knife cone biopsy of the cervix.

Woodford HD, Poston W, Elkins TE.

J Reprod Med 1986 Oct;31(10):951-3 Abstract quote

Eight patient records were reviewed following cold knife conization in which frozen section diagnosis was utilized to aid the surgeon in formulating appropriate therapy after conization.

Two patients were diagnosed as having microinvasive squamous cell carcinoma on final pathology when frozen sections were read as showing no invasion. Furthermore, in the 51 instances in which a degree of cervical dysplasia was determined from frozen sections, 14 discrepancies were noted on final pathology (27%).

Such discrepancies may lead to unnecessary hysterectomies performed for cervical dysplasia that is easily treated with outpatient office procedures, especially when cold knife conization and frozen section diagnosis are performed without prior colposcopy and biopsy.

Evaluating cervical cone biopsy specimens with frozen sections at hysterectomy.

Neiger R, Bailey SA, Wall AM 3rd, Jennings JB, Gallup DG.

Department of Obstetrics and Gynecology, Memorial Medical Center, Inc., Savannah, GA 31404.

J Reprod Med 1991 Feb;36(2):103-7 Abstract quote

Frozen section evaluations of cervical cone biopsy specimens were performed at the time of hysterectomy to exclude invasive cervical cancer.

During a two-year period we prospectively evaluated 43 cone biopsy specimens. We found all the diagnoses made with frozen sections to be accurate when compared with prospective permanent sections, and all patients received appropriate therapy. Thirty-eight cases showed no evidence of invasion. Two patients had invasive squamous cell cervical cancer, one had invasive cervical adenocarcinoma extending to the endometrial cavity, and two had microinvasion. All invasive cancers were diagnosed correctly with frozen sections and confirmed with permanent sections. When hysterectomy immediately followed conization, no complications occurred, and no significant increase in blood loss was noted.

We found frozen section evaluation of a cone biopsy specimen at the time of hysterectomy to be a reliable procedure that saves time, eliminates the risk of additional anesthesia and decreases patients' costs.

Cervical conization with frozen section before planned hysterectomy.

Hoffman MS, Collins E, Roberts WS, Fiorica JV, Gunasekaran S, Cavanagh D.

Department of Obstetrics and Gynecology, University of South Florida College of Medicine.

Obstet Gynecol 1993 Sep;82(3):394-8 Abstract quote

OBJECTIVE: To report our institutional experience with the accuracy and usefulness of cervical conization with frozen section before planned hysterectomy.

METHODS: One hundred fifty-nine patients who planned to have hysterectomies for or with a concomitant diagnosis of cervical intraepithelial neoplasia (CIN) underwent preliminary cone biopsies with frozen section. The frozen and permanent pathologic diagnoses were compared retrospectively. Detailed analysis was directed at the indications for cone biopsy and the patients who were found to have invasive cancer.

RESULTS: Among 108 patients with negative specimens or CIN, the frozen section was accurate within one degree of CIN in 106. Cone biopsy for a positive endocervical curettage, unsatisfactory colposcopy, or discrepant cytology did not contribute to the diagnosis of invasive cancer but did lead to an ultimate diagnosis of CIN III. One of 12 women with a frozen-section diagnosis of microinvasion had deeper invasion on permanent sections.

CONCLUSIONS: Frozen-section evaluation of a cone biopsy carries a degree of accuracy that enables the surgeon to make an immediate decision about definitive therapy. Exact indications, use during pregnancy, and accuracy for the diagnosis of microinvasion require further study.

Value of endocervical margin examination of conization specimens. Prospective study conducted on 150 patients [Article in French]

Rojat-Habib MC, Cravello L, Bretelle F, Roger V, Liprandi A, de Burtel I, d'Ercole C, Pellissier JF, Blanc B.

Service d'anatomie-pathologique et de neuropathologie, hopital de la Timone, Marseille, France.

Gynecol Obstet Fertil 2000 Jul-Aug;28(7-8):518-25 Abstract quote

OBJECTIVE: To assess the usefulness of frozen sections (FS) on endocervical margin in surgical conization or loop electrosurgical specimens.

MATERIAL AND METHODS: In a prospective study, 150 patients were treated from October 1995 to December 1997: 69 cases without FS, 81 cases with FS. CIN on frozen section resulted in an immediate additional resection.

RESULTS: In the group without FS, 13 patients had involved endocervical margin by high-grade CIN (18.8%). Frozen section was impossible in a conization specimen that was too short. FS revealed 64 normal glandular epitheliums, seven squamous metaplasias in which two lesions were under-evaluated (being in fact CIN on permanent sections), eight high-grade CIN followed by additional resection in six cases and two invasive carcinomas. Endocervical margin on additionals section were always free of disease. The rate of failure was 2.6% among 77 cases. This rate corresponded to two under-evaluations. Invasive carcinoma and CIN without additional resection were excluded because frozen section only allowed a peroperative diagnosis. The average height of the cone and the rate of complications were similar. Repeat surgery was necessary in nine cases in the group without frozen section, in which five showed residual lesions, absent in the other group.

CONCLUSION: The ultimate histological interpretation was never difficult after frozen section. This method permits reduction of cases with involved cone margin and residual lesions and, despite some limitations, it may be useful for surgical management


A controlled trial of a human papillomavirus type 16 vaccine.

Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB, Chiacchierini LM, Jansen KU; Proof of Principle Study Investigators.

Department of Epidemiology, University of Washington, Seattle, USA.

N Engl J Med 2002 Nov 21;347(21):1645-51 Abstract quote

BACKGROUND: Approximately 20 percent of adults become infected with human papillomavirus type 16 (HPV-16). Although most infections are benign, some progress to anogenital cancer. A vaccine that reduces the incidence of HPV-16 infection may provide important public health benefits.

METHODS: In this double-blind study, we randomly assigned 2392 young women (defined as females 16 to 23 years of age) to receive three doses of placebo or HPV-16 virus-like-particle vaccine (40 microg per dose), given at day 0, month 2, and month 6. Genital samples to test for HPV-16 DNA were obtained at enrollment, one month after the third vaccination, and every six months thereafter. Women were referred for colposcopy according to a protocol. Biopsy tissue was evaluated for cervical intraepithelial neoplasia and analyzed for HPV-16 DNA with use of the polymerase chain reaction. The primary end point was persistent HPV-16 infection, defined as the detection of HPV-16 DNA in samples obtained at two or more visits. The primary analysis was limited to women who were negative for HPV-16 DNA and HPV-16 antibodies at enrollment and HPV-16 DNA at month 7.

RESULTS: The women were followed for a median of 17.4 months after completing the vaccination regimen. The incidence of persistent HPV-16 infection was 3.8 per 100 woman-years at risk in the placebo group and 0 per 100 woman-years at risk in the vaccine group (100 percent efficacy; 95 percent confidence interval, 90 to 100; P<0.001). All nine cases of HPV-16-related cervical intraepithelial neoplasia occurred among the placebo recipients.

CONCLUSIONS: Administration of this HPV-16 vaccine reduced the incidence of both HPV-16 infection and HPV-16-related cervical intraepithelial neoplasia. Immunizing HPV-16-negative women may eventually reduce the incidence of cervical cancer.

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