The use of p16INK4A immunocytochemistry in “Atypical squamous cells which cannot exclude HSIL” compared with “Atypical squamous cells of undetermined significance” in liquid‐based cervical smears

Even though p16^INK4a (p16) immunocytochemistry has proven a useful accessory tool verifying the identification of atypical squamous cells of undetermined significance (ASC‐US) categorized smears, the procedure still has limitations. To date few studies examining the usefulness of p16 immunocytochemistry in atypical squamous cells which cannot exclude HSIL (ASC‐H), compared with ASC‐US in liquid‐based cervical smears. Therefore, we examined the correlation of p16 immunocytochemical staining with follow‐up biopsy results on ASC‐H categorized smears and compared the data with those classified as ASC‐US on 105 liquid‐based cytology samples. We found no statistical significance in the p16 expression of ASC‐US smears and the presence of squamous intraepithelial lesions (SIL) in follow‐up biopsies (p = 0.546). However, p16 expression did significantly correlate with the presence of SIL (p = 0.002) in ASC‐H smears. There was a statistically significant relationship between p16 expression and presence of high grade squamous intraepithelial lesions (HSIL) or more on the follow‐up biopsies in both ASC‐US (p = 0.012) and ASC‐H (p < 0.001) categorized smears. In ASC‐US categorized smears, there was no statistical significance between p16 expression and the HR‐HPV viral load (p = 0.091). But there was a statistical significance between p16 expression and the HR‐HPV viral load (p < 0.001) in ASC‐H categorized smears. Our results indicate that p16 immunostaining is a much better useful marker for HR‐HPV infection and detection of SIL in ASC‐H categorized smears compared to those defined as ASC‐US. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

CINtec® PLUS dual immunostain: A triage tool for cervical pap smears with atypical squamous cells of undetermined significance and low grade squamous intraepithelial lesion

ASC and LSIL comprise the majority of abnormal Pap smears. Currently, high‐risk human papillomavirus testing is utilized to triage women with ASC for colposcopy; however, no cost effective triage method is available for LSIL. p16 and Ki‐67 have each been shown to be good biomarkers for high grade cervical intraepithelial neoplasia (HG CIN).We evaluated the role of the CINtec® PLUS p16/Ki‐67 dual immunostain as a marker for underlying (U) or subsequent (S) HG CIN. One hundred and eighty eight cervical SurePath Pap smears with histological and/or cytological follow‐up were retrieved from our departmental files. The Pap stained slides were destained and then immunostained utilizing the CINtec® PLUS dual staining reagent kit. Results of the dual stain were correlated with follow‐up diagnoses. Sensitivity, specificity, and positive and negative predictive values of CINtec® PLUS for U or S HG CIN were compared with those of HR HPV testing and with p16 and Ki‐67 immunostaining alone. The sensitivity of CINtec® PLUS for U or S HG CIN was 91% in the ASC group and 100% in the LSIL group, while the corresponding specificities were 61 and 43%, respectively. The sensitivity and specificity of CINtec® PLUS for U or S HG CIN in both groups combined were 97 and 53%, respectively. CINtec® PLUS was more specific than HR HPV testing and Ki‐67 and p16 immunostains alone in detecting an U or S HG CIN. CINtec® PLUS is a helpful adjunct in identifying U or S HG CIN when applied to SurePath Pap smears with ASC or LSIL. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

Atypical squamous cells,cannot exclude high‐grade squamous intraepithelial lesion: Diagnostic features in surepath™ cervical samples

This study was undertaken to identify the situations in which a diagnosis of “Atypical squamous cells, cannot exclude a high‐grade squamous intraepithelial lesion (ASC‐H)” is offered in SurePath? cervical samples and to identify cytological criteria helpful in predicting high‐grade disease. 2,335 (3.4%) SurePath samples reported as atypical squamous cells (ASC) over a period of 2 years, including 1,112 cases with known hrHPV status were retrieved. 105/1,112 cases were categorized into ASC‐H, and slides were available for review in 88/105 cases. These 88 samples were divided into two categories based on follow‐up histological outcome and hrHPV status–category A: cases with CIN2+ lesions on follow‐up (n = 48) and category B: cases with ≤CIN1 lesions or hrHPV negative status (n = 40). 78% (82/105) cases of ASC‐H tested positive for hrHPV. Overall CIN2+ lesions were found in 50.3% (53/105) cases. Of 88 cases reviewed, HCGs were noted in 56.3% (27/48) cases in category A and 75% (30/40) cases in category B. Dispersed metaplastic cells and scattered small atypical cells were seen in 37.5% (18/48) cases in category A and 12.5%(5/40) in category B. The majority of cases with dispersed atypical cells had <20 cells/sample and cases with HCGs had <10 HCGs per sample. The majority of the cases reported as ASC‐H contained HCGs. Of these groups with nuclear crowding, disorganization and those with steep edges (“blocks”) are likely to predict high‐grade disease. The samples with only dispersed atypical cells had <20 cells/sample in majority of cases. In these, a disproportionate andespecially high nuclear: cytoplasmic ratio and irregular chromatin were the most useful features in predicting high‐grade disease. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

Management of atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions of the uterine cervix with human papilloma virus infection among young women aged less than 25 years

Current ASCCP guidelines recommend repeat cytology 12 months after HPV‐positive results in women aged 21–24 years with either atypical squamous cells of undetermined significance (ASCUS) or a low‐grade squamous intraepithelial lesion (LSIL). The purpose of this study was to validate an algorithm in such women with ASCUS or LSIL. A multicenter cross‐sectional study was carried out at three academic hospitals involving 40,847 Korean women who underwent cervical cancer screening with cytology and HPV testing with or without subsequent colposcopic biopsies between January 2007 and December 2013. Among a total of 3,193 women with available histopathology data, 762 women with ASCUS and 758 with LSIL were HPV‐positive. Among HPV‐positive women with ASCUS, 38.5% of women aged 21–24 years had ≥CIN2, compared to 20.8% of women aged 30–65 years and 21.1% of the total women. Among HPV‐positive women with LSIL, 25.8% aged 21–24 years had ≥CIN2, compared to 21.2% of women aged 30–65 years and 21.9% of the total women. In HPV‐positive women with ASCUS/LSIL aged less than 25 years, the prevalence of ≥CIN2 lesions was 34.5%, which was significantly higher than that (21.0%) in women aged ≥25 years. The risk of ≥CIN2 lesions in HPV‐positive Korean women aged 21–24 years with ASCUS or LSIL was not lower than that in older women. Colposcopic examination should be considered for management of HPV‐positive young women with ASCUS or LSIL. Diagn. Cytopathol. 2016;44:959–963. © 2016 Wiley Periodicals, Inc.     

An evaluation of the diagnostic and prognostic significance of p16INK4a/p21WAF1/Cip1 immunostaining in squamous intraepithelial lesions of the uterine cervix using liquid‐based cytology specimens

Human papillomavirus (HPV) infection frequently causes squamous intraepithelial lesions (SIL) of the uterine cervix and consequently gives rise to squamous cell carcinoma. It is therefore important to identify cases that potentially develop higher grades of SIL at an early stage of the disease. In this study, we thus investigated whether immunocytochemistry for p21^WAF1/Cip1 and p16^INK4a could be applicable in the diagnosis and the prognostic prediction of SIL in combination with genomic analyses of HPV. The genomic analysis of high‐risk HPV (hrHPV), which was done by reversed dot blotting and by in situ hybridization, and immunocytochemistry were performed on liquid‐based cytological specimens. A cross‐sectional study comprising 145 cases of NILM, ASC‐US, LSIL, and HSIL indicated that the incidence of the positive cases for p16^INK4a and p21^WAF1/Cip1 and hrHPV increased with the grade of SIL. A double positive status for p16^INK4a and p21^WAF1/Cip1 was a significant discriminator between HSIL and LSIL/NILM, even when applied in conjunction with the genomic test for hrHPV (P = 0.006 by logistic regression analysis). However, a prospective study employing 61 NILM/ASC‐US cases, revealed that the p16^INK4a/p21^WAF1/Cip1 immunostaining was not a significant predictor for the progression of SIL, whereas the cytological diagnosis (NILM vs. ASC‐US) and the infection status of hrHPV conferred significant effects on the prognosis. Immunostaining of p16^INK4a and p21^WAF1/Cip1 provides additional information on the cytological diagnosis of SIL. A further analysis using a larger population is warranted to obtain a conclusive result regarding the prognostic significance of p16^INK4a/p21^WAF1/Cip1 immunocytochemistry in the diagnosis of SIL. Diagn. Cytopathol. 2014;42: 125–133. © 2013 Wiley Periodicals, Inc.     

Distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in Canada

Infection with high‐risk human papillomavirus (HPV) causes cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). The distribution of HPV types in cervical diseases has been previously described in small studies for Canadian women. The prevalence of 36 HPV genotypes in 873 women with CIN and 252 women with ICC was assessed on cervical exfoliated cells analyzed with the Linear Array (Roche Molecular System). HPV16 was the most common genotype in CIN and ICC. The seven most frequent genotypes in order of decreasing frequency were HPV16, 51, 52, 31, 39, 18, and 56 in women with CIN1, HPV16, 52, 31, 18, 51, 39, and 33 in women with CIN2, HPV16, 31, 18, 52, 39, 33, and 58 in women with CIN3, and HPV16, 18, 45, 33, 31, 39, and 53 in women with ICC. HPV18 was detected more frequently in adenocarcinoma than squamous cell carcinoma (P = 0.013). Adjustment for multiple type infections resulted in a lower percentage attribution in CIN of HPV types other than 16 or 18. The proportion of samples containing at least one oncogenic type was greater in CIN2 (98.4%) or CIN3 (100%) than in CIN1 (80.1%; P < 0.001 for each comparison). Multiple type infections were demonstrated in 51 (20.2%) of 252 ICC in contrast to 146 (61.3%) of 238 women with CIN3 (P < 0.001). Adjusting for multiple HPV types, HPV16 accounted for 52.1% and HPV18 for 18.1% of ICCs, for a total of 70.2%. Current HPV vaccines should protect against HPV types responsible for 70% of ICCs in Canadian women. J. Med. Virol. 83:1034–1041, 2011. © 2011 Wiley‐Liss, Inc.     

Clinical significance of atypical squamous cells cannot exclude high grade squamous intraepithelial lesion with histologic correlation—: A 9‐Year experience

Atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion (ASC‐H) is a recognized category in the 2001 Bethesda Nomenclature System for cervical cytology. Although current ASCCP guidelines recommend colposcopic follow‐up, more recent studies are suggesting prior triage for HPV‐DNA analysis. We report on our experience at the University of Wisconsin Hospital and Clinics. From January 1, 2003 through December 31, 2011 (9‐y), the cytopathology laboratory processed 109,424 Pap Tests, of which 281 (0.26%) were diagnosed as ASC‐H. Tissue follow‐up was available in 181 (64%) of these cases, of which 45 (25%) were negative/cervicitis, 41 (23%) were CIN 1, 36 (20%) were CIN 2 and 59 (32%) were CIN 3. Stratification by age groups showed a higher percentage of high grade (CIN 2+) lesions (65%) in the premenopausal age group as compared with high grade lesion (35%) in the postmenopausal age group, whereas negative/CIN1 biopsies were more common in postmenopausal (65%) as compared to premenopausal (44%) women. Our data support the use of colposcopy in the management of women with ASC‐H on Pap Tests. However, in the older age group, prior HPV‐DNA testing may be of benefit to better identify those women at risk for high grade lesions. Diagn. Cytopathol. 2013;41:943–946. © 2013 Wiley Periodicals, Inc.     

p16(INK4A) is a surrogate biomarker for a subset of human papilloma virus-associated dysplasias of the uterine cervix as determined on the Pap smear

Recently, p16(INK4A) has been identified as a biomarker for human papilloma virus (HPV)-induced dysplastic lesions of the cervix and it has been suggested that it may be a useful diagnostic aid for these lesions. This study therefore was performed to determine the utility of p16 expression in a series of Papanicolaou (Pap) smears collected in liquid medium and to determine its benefit, if any, over HPV testing. One hundred seven cases, including 23 negative cases, 34 with low-grade squamous intraepithelial lesion (LSIL), 16 with high-grade squamous intraepithelial lesion (HSIL), 29 with atypical squamous cells of uncertain significance (ASC-US), and 5 cases with ASC suspicious for HSIL (ASC-H), were evaluated for both p16 expression and HPV DNA. We observed p16 expression in only 36% of all cases with abnormal cytology (30/84) and in 40% of all cases associated with high-risk HPV. The highest rate of positivity (80%) and the highest levels of expression (more than three to five positive cells/10x field) were seen in HSIL. Similar results were observed with ASC-H cases. This suggests that in equivocal cases, p16 may be used for confirmation of the diagnosis. On the other hand, p16 positivity was noted in only 21% of LSIL and ASC-US cases. This raises the interesting possibility, given that only a minority of LSIL cases progress on to higher-grade lesions, that p16 might be useful for triaging these patients for closer follow-up and/or further evaluation. Additional studies are required for confirmation.     

Oral antibodies to human papillomavirus type 16 in women with cervical neoplasia

This study investigated the relationship between human papillomavirus type 16 (HPV-16) antibodies detected in oral fluid from women with cervical neoplasia, their HPV-16 antibody seroprevalence, and their cervical HPV-16 DNA presence. Cervical HPV-16 DNA was detected by polymerase chain reaction in 43.2% (35/81) of these women. The prevalence of IgG and IgA antibodies to HPV-16 virus-like particles (VLP-16) in oral fluid and was investigated by enzyme-linked immunosorbent assay. Anti-VLP-16 IgA antibodies were detected in oral fluid from 54.3% (44/81) of women with cervical neoplasia, compared with 8% (3/36) in controls (P = 0.000002). Anti-VLP-16 IgG was detected in oral fluid from 43.2.9% (25/72) and 13.3% (4/30; P = 0.029), respectively. Women who were HPV-16 DNA positive at their cervical lesion, displayed an oral fluid anti-VLP-16 IgA prevalence of 60.7% (17/28) and HPV-16 DNA negative women an oral fluid anti-VLP-16 IgA prevalence of 50% (20/40; P = 0.38). Oral fluid anti-VLP-16 IgG prevalence in HPV-16 DNA positive women was 28.6% (8/28) compared with 40% (16/40) in oral fluid from HPV-16 DNA negative women (P = 0.3). Amongst HPV-16 DNA positive women, the anti-VLP-16 IgG seroprevalence was 75% (21/28) and IgA seroprevalence 35.7% (10/28) and for the HPV-16 DNA negative women these values were 60% (24/40) and 32.5% (13/40), respectively. Oral IgA antibody testing proved no more sensitive than serum antibody detection for the determination of HPV infection but could be useful as a non-invasive screening method for women with cervical neoplasia and for estimating the mucosal antibody response to HPV vaccines.     

p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection

Houghton O, Jamison J, Wilson R, Carson J & McCluggage W G
(2010) Histopathology 57, 342–350
p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection Aims: The association between human papillomavirus (HPV) and cervical carcinoma is well known, with HPV being identifiable in almost all cervical squamous carcinomas and most adenocarcinomas. However, the prevalence of HPV in unusual morphological types of cervical adenocarcinoma has not been investigated extensively. The aim was to determine HPV status in a series of primary cervical adenocarcinomas, enriched for unusual morphological types. The relationship between HPV and p16 immunoreactivity in these neoplasms was also investigated, as it is generally assumed that in cervical neoplasms diffuse p16 expression is predictive of the presence of high‐risk HPV. Methods and results: Sixty‐three cervical adenocarcinomas, comprising those of usual type (n = 43), minimal deviation type (n = 4), gastric type (n = 3), intestinal type (n = 3), mesonephric type (n = 3), clear cell type (n = 4), serous type (n = 2) and hepatoid type (n = 1) underwent linear array HPV genotyping and immunohistochemistry for p16. Overall, HPV was identified in 32 of 56 cases (57%) in which sufficient DNA was present for analysis. The most common HPV types were 16 and 18, with these being identified in 20 and 18 cases, respectively, either alone or in combination. Seventy‐eight per cent of usual‐type adenocarcinomas were HPV‐positive, as was the single serous carcinoma in which there was sufficient DNA for analysis. In contrast, all minimal deviation adenocarcinomas and those of gastric, intestinal, mesonephric and clear cell types were HPV‐negative, as was the single hepatoid carcinoma. All usual‐type adenocarcinomas exhibited p16 immunoreactivity (diffuse staining in all but one case), as did 11 of 20 of those of unusual morphological type (five focal, six diffuse). Conclusions: Most, but not all, cervical adenocarcinomas of usual type contain HPV, but those of unusual morphological type are almost always HPV‐negative. This has implications for the efficacy of HPV vaccination in the prevention of cervical adenocarcinoma. A significant proportion of cervical adenocarcinomas are p16‐positive in the absence of HPV, illustrating that in these neoplasms diffuse p16 immunoreactivity is not a reliable surrogate marker of the presence of high‐risk HPV.     

p16INK4a expression as a potential marker of low‐grade cervical intraepithelial neoplasia progression

To evaluate p16^INK4a immunoexpression in CIN1 lesions looking for differences between cases that progress to CIN2/3 maintain CIN1 diagnosis, or spontaneously regress. Seventy‐four CIN1 biopsies were studied. In the follow‐up, a second biopsy was performed and 28.7% showed no lesion (regression), 37.9% maintained CIN1, and 33.4% progressed to CIN2/3. Immunostaining for p16^INK4a was performed in the first biopsy and it was considered positive when there was strong and diffuse staining of the basal and parabasal layers. Pearson's chi‐square was used to compare the groups (p ≤ 0.05). The age of the patients was similar. There was no significant difference in p16^INK4a immunoexpression in the groups, however, statistical analyses showed a significant association when only the progression and regression groups were compared (p = 0.042). Considering p16^INK4a positivity and the progression to CIN2/3, the sensitivity, specificity, positive, and negative predictive values in our cohort were 45%, 75%, 47%, and 94%, respectively. We emphasize that CIN1 with p16^INK4a staining was associated with lesion progression, but the sensitivity was not high. However, the negative predictive value was more reliable (94%) and p16^INK4a may represent a useful biomarker that can identify CIN1 lesions that need particular attention, complementing morphology.     

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.     

宫颈上皮内瘤变中p16INK4A和Ki-67的表达

摘要：目的探讨p16^INK4A、Ki-67和HPV抗原表达及高危型HPV（HR—HPV）检测在宫颈上皮内瘤变（CIN）病理诊断中的意义。方法选取宫颈活检病理确诊为CIN的组织蜡块101例,重新切片,应用免疫组化两步法检测p16^INK4A、Ki-67和HPV抗原表达,并取正常宫颈组织50例进行对比研究。同时,应用第二代杂交捕获法对其中25例CIN组织样品进行HR—HPV检测。结果p16^INK4A蛋白表达水平在CINI、CINⅡ、CINⅢ级之间差异有非常显著性（P〈0．001）,其表达水平随着CIN级别的增高而增加,呈现良好的线性相关性（P〈0．001）;Ki-67蛋白表达阳性细胞多少与CIN分级之间无显著相关性（P〉0．05）,但其在宫颈鳞状上皮中的位置分布与CIN级别之间却有显著相关性（P〈0．05）;HPV抗原免疫组化染色阳性反应仅呈现于CIN鳞状上皮表层挖空细胞内,其阳性率在不同级别CIN之间的差异无统计学意义（P〉0．05）;HR—HPV在CINI、CINⅡ和CINⅢ级的检出率分别为81．8％、80．0％和100．0％,但其检出率在不同级别CIN之间差异也无显著性（P〉0．05）。结论p16^INK4A和Ki-67染色对CIN的病理诊断和分级具有一定诊断价值,对于CINI级形态结构不典型的病例,HR—HPV的检测结果对病理诊断有辅助意义。     

Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion

This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.     

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4(+) and CD8(+) T cells from the cervix responded to the HPV-16 antigens and that interferon-gamma (IFN-gamma) production was HPV type-specific. Comparing HPV-specific T-cell IFN-gamma responses at the cervix with those in the blood, we found that while CD4(+) and CD8(+) T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-gamma responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4(+) and CD8(+) T-cell IFN-gamma responses to E7 were of similar magnitude in both compartments and CD8(+) responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.