Triaging HPV‐positive women with normal cytology by p16/Ki‐67 dual‐stained cytology testing: Baseline and longitudinal data

Primary human papillomavirus (HPV)‐based screening results in a 2–5% lower specificity for cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) compared to Pap cytology. To identify HPV‐positive women with CIN2+, we retrospectively evaluated the cross‐sectional and longitudinal performance of p16/Ki‐67 dual‐stained cytology in HPV‐positive women with normal cytology participating in population‐based cervical screening. Conventional Pap cytology specimens of 847 of these women derived from the VUSA‐Screen study were dual‐stained for p16/Ki‐67. Cross‐sectional clinical performance in detecting CIN3 or worse (CIN3+), and CIN2+ was compared to that of baseline HPV genotyping. Moreover, 5‐year cumulative incidence risks (CIR) for CIN3+ (CIN2+) were determined. The sensitivity of p16/Ki‐67 dual‐stained cytology for CIN3+ (CIN2+) was 73.3% (68.8%) with a specificity of 70.0% (72.8%). HPV16/18 genotyping showed a sensitivity for CIN3+ (CIN2+) of 46.7% (43.8%), with a specificity of 78.3% (79.4%). The 5‐year CIR for CIN3+ in HPV‐positive women with normal cytology was 6.9%. Testing these women with p16/Ki‐67 dual‐stained cytology resulted in a significantly lower CIN3+ 5‐year CIR of 3.3% (p = 0.017) in case of a negative test result. A negative HPV16/18 genotyping test result also led to a lower 5‐year CIN3+ CIR of 3.6%. p16/Ki‐67 dual‐stained cytology detects more than 70% of underlying CIN3+ lesions in HPV‐positive women with normal cytology at baseline and is therefore suitable for triaging these women to colposcopy. Furthermore, the CIN3+ 5‐year CIR of 3.3% after a negative dual‐stain result is significantly lower compared to the 5‐year CIR of 6.9% in women without p16/Ki‐67 dual‐stained cytology triage.     

Evaluation of p16INK4a/Ki‐67 dual stain in comparison with an mRNA human papillomavirus test on liquid‐based cytology samples with low‐grade squamous intraepithelial lesion

BACKGROUND:

The objective of the current study was to investigate the clinical performance of detecting high‐grade lesions with the CINtec PLUS p16^INK4a/Ki‐67 dual stain and the APTIMA human papillomavirus (HPV) Assay in a cohort of women with low‐grade squamous intraepithelial lesion (LSIL) cytology. The authors also assessed the reproducibility of the evaluation of immunocytochemical staining.

METHODS:

The 2 tests were performed on liquid‐based residual material from 469 women with LSILs. The samples had at least 5 years of follow‐up and the gold standard used was high‐grade cervical intraepithelial neoplasia (CIN2+/CIN3+) proven on histology.

RESULTS:

Approximately 69% of all the women included in the study had a positive test for HPV mRNA and 56% was positive for the dual stain. The 2 tests demonstrated high sensitivities. When examining the specificities, the APTIMA HPV Assay performed with significantly lower values than the CINtec PLUS test. For patients with CIN2+, the APTIMA HPV Assay had a specificity of 36.1% versus 51.3% for the CINtec PLUS test, and for women with CIN3+, the specificity was 33.8% versus 48.2%, respectively. The difference was even more pronounced when analyzing women aged < 30 years separately. The kappa values between the 3 observers in scoring the dual stain ranged from 0.43 to 0.49 and improved in a second evaluation round to values ranging from 0.50 to 0.66.

CONCLUSIONS:

The CINtec PLUS p16^INK4a/Ki‐67 dual‐staining test in LSIL cytology samples demonstrated high sensitivity that was similar to that of the APTIMA HPV Assay in the detection of underlying high‐grade disease but with enhanced specificity, especially among women aged < 30 years. The kappa value for the evaluation of the CINtec PLUS dual‐staining test was moderate but could be improved through training. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.     

The role of human papillomavirus type 16/18 genotyping in predicting high‐grade cervical/vaginal intraepithelial neoplasm in women with mildly abnormal Papanicolaou results

BACKGROUND:

The authors compared the predictive value of type 16 and/or 18 human papillomavirus (HPV) versus non‐16/18 HPV types for high‐grade (grade ≥2) cervical neoplasm/vaginal intraepithelial neoplasm and carcinoma (CIN/VAIN2+) in women with mildly abnormal Papanicolaou (Pap) results (ie, atypical squamous cells of undetermined significance [ASCUS] or low‐grade squamous epithelial lesion [LSIL]).

METHODS:

The authors retrospectively selected Pap specimens with HPV testing results obtained from 243 women (155 with ASCUS and 88 with LSIL Pap results) in their Department of Pathology. HPV genotyping was performed using the EasyChip HPV blot assay. The Pap specimens with HPV16/18 and non‐16/18 HPV types were compared with follow‐up biopsy results. Follow‐up duration ranged from 1 month to 58 months (mean, 26 months).

RESULTS:

In total, 58 of 155 specimens (37%) that had ASCUS and 29 of 88 specimens (33%) that had LSIL were positive for HPV16/18. CIN/VAIN2+ biopsies were identified in 43 of 155 women (28%) with ASCUS and in 28 of 88 women (32%) with LSIL. Women with ASCUS and HPV16/18 had a significantly higher rate (43%) of CIN/VAIN2+ than women with ASCUS and non‐16/18 HPV types (19%; P = .003; odds ratio, 3.10; 95% confidence interval, 1.48‐6.53). There was no statistically significant difference in the rate of CIN/VAIN2+ between women who had LSIL and HPV16/18 (45%) and those who had LSIL and non‐16/18 HPV types (29%; P = .16; odds ratio, 1.96; 95% confidence interval, 0.77‐4.97).

CONCLUSIONS:

HPV genotyping for HPV16/18 improved risk assessment for women with ASCUS Pap results and may be used to predict the risk of CIN/VAIN2+ to better guide follow‐up management. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.     

Immunocytochemical colocalization of P16(INK4a) and Ki-67 predicts CIN2/3 and AIS/adenocarcinoma

BACKGROUND:

Although previous studies have shown that p16^INK4a and Ki‐67 are sensitive and specific markers for high‐grade lesions (≥CIN2) on cervical biopsies, limited information is available regarding the performance of a dual‐staining approach as a diagnostic adjunct in cervical cytology. We evaluated a dual p16^INK4a/Ki‐67 immunocytochemistry (ICC) assay to determine its sensitivity and specificity versus that of high‐risk HPV (HR‐HPV) in a US‐based pilot cytology study.

METHODS:

ThinPrep specimens from 122 cervical cytology specimens encompassing 23 negative (NILM), 20 ASC‐US, 22 LSIL, 17 ASCH, 22 HSIL, and 18 AGC cases were processed for multiplexed ICC staining using a CINtec Plus Kit. Dual‐positive assay results were defined based on the detection of 1 or more epithelial cells that were stained for both p16^INK4a and Ki‐67 without regard to cellular morphology. HR‐HPV testing was performed by multiplex PCR with capillary electrophoresis genotyping.

RESULTS:

Dual staining for p16^INK4a and Ki‐67 was frequently detected in HSIL and AGC but was rarely detected in NILM cases. The HR‐HPV assay showed a sensitivity of 76.2% and a specificity of 55.8% for the detection of clinically significant cervical squamous or endometrial lesions. In contrast, the colocalization of p16^INK4a plus Ki‐67 maintained a high sensitivity of 81.8% and improved specificity to 81.8% for biopsy‐confirmed CIN2/3, endocervical adenocarcinoma, or endometrial adenocarcinoma.

CONCLUSIONS:

Dual staining for p16^INK4a/Ki‐67 immunocytochemistry dramatically increased specificity and maintained high‐level sensitivity for the diagnosis of CIN2/3 or glandular lesions compared with PCR‐based testing for HR‐HPV. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.     

Triaging borderline/mild dyskaryotic Pap cytology with p16/Ki-67 dual-stained cytology testing: cross-sectional and longitudinal outcome study

Background:

Women with borderline/mildly dyskaryotic (BMD) cytology smears are currently followed up with repeat testing at 6 and 18 months. The objective of this study is to analyse the cross-sectional and longitudinal performance of p16/Ki-67 dual-stained cytology for the detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) and CIN2+ in women with BMD, and to compare the results with baseline human papillomavirus (HPV) testing.

Methods:

Conventional Pap cytology specimens of 256 women with BMD were dual stained for p16/Ki-67 retrospectively, and compared with baseline HPV results and long-term follow-up results.

Results:

p16/Ki-67 dual-stained cytology showed a sensitivity of 100%, a specificity of 64.4% and a negative predictive value (NPV) of 100.% for CIN3+. Human papillomavirus testing demonstrated similar sensitivity (96.3%), and NPV (99.1%), but a significantly lower specificity (57.6% P=0.024) for CIN3+. Sensitivity, specificity and NPV for CIN2+ of dual-stained cytology were 89.7%, 73.1% and 95.1%, respectively, which was similar when compared with HPV testing. Dual-stained cytology showed a significant lower referral rate than HPV testing (43.6% vs 49.1% P=0.043). During long-term follow-up, no CIN3+ lesions developed in HPV-positive, dual-stained negative women.

Conclusions:

Comparable sensitivity and NPV of dual-stained cytology for CIN3+, combined with a significantly higher specificity, makes p16/Ki-67 dual-stained cytology a viable alternative to HPV testing for triaging BMD.     

Good performance of p16/ki‐67 dual‐stained cytology for surveillance of women treated for high‐grade CIN

Women treated for high‐grade cervical intraepithelial neoplasia (CIN) are at risk of recurrent CIN Grade 2 or worse (rCIN2+). Currently, posttreatment monitoring is performed using cytology or cytology/high‐risk (hr)HPV cotesting. This study aimed to evaluate the performance of p16/Ki‐67 dual‐stained cytology (p16/Ki‐67) for posttreatment monitoring. Three hundred and twenty‐three women treated for high‐grade CIN in the SIMONATH study underwent close surveillance by cytology, hrHPV and DNA methylation marker testing up to 12 months posttreatment. Histological endpoints were ascertained by colposcopy with biopsy at 6 and/or 12 months. p16/Ki‐67 dual‐staining was performed on residual liquid‐based cytology samples obtained at, or shortly before biopsy collection. Clinical performance estimates of cytology, hrHPV, p16/Ki‐67 testing and combinations thereof for the detection of rCIN2+ were determined and compared to each other. Sensitivity of p16/Ki‐67 for rCIN2+ (69.2%) was nonsignificantly lower than that of cytology (82.1%; ratio 0.84, 95% CI: 0.71–1.01), but significantly lower than that of hrHPV testing (84.6%; ratio 0.82, 95% CI: 0.68–0.99). Specificity of p16/Ki‐67 for rCIN2+ (90.4%) was significantly higher compared to both cytology (70.8%; ratio 1.28, 95% CI: 1.19–1.37) and hrHPV testing (76.2%; ratio 1.19, 95% CI: 1.12–1.26). Overall, hrHPV testing showed very high sensitivity, along with a good specificity. When considering cotesting, combined p16/Ki‐67/hrHPV testing showed rCIN2+ sensitivity comparable to cytology/hrHPV cotesting (87.2% vs. 89.7%; ratio 0.97, 95% CI: 0.92–1.03), but with significantly increased specificity (74.2% vs. 58.1%; ratio 1.28, 95% CI: 1.19–1.38). Thus, when considered in combination with hrHPV, p16/Ki‐67 might be an attractive approach for surveillance of women treated for high‐grade CIN.     

Triage of women with ASCUS and LSIL cytology

BACKGROUND.

The identification of a small percentage of high‐grade cervical intraepithelial neoplasias (HGCIN) among patients with minor cytological abnormalities (atypical squamous cells of undetermined significance [ASCUS] and/or low‐grade squamous intraepithelial lesions [LSIL] group) is a major problem in cytology‐based cervical cancer screening. The authors investigated the efficacy of p16^INK4a as a biomarker to identify samples of patients with HGCIN among those with an ASCUS or LSIL result in Papanicolaou cytology.

METHODS.

Consecutive liquid‐based cytology specimens of 137 ASCUS and 88 LSIL results were selected from gynecologists who adopted a triage regimen with biopsy under colposcopy 2 months later, independent of the p16^INK4a result. p16^INK4a stained slides were prepared and independently read by 2 observers, who used a recently described score to categorize p16^INK4a stained squamous cells. The endpoint of the study was detection of a biopsy‐confirmed HGCIN.

RESULTS.

The overall sensitivity and specificity of p16^INK4a positive cells with a nuclear score >2 for diagnosis of HGCIN in ASCUS and LSIL cases combined was 96% and 83%, respectively. The sensitivity and specificity in the ASCUS group was 95% and 84%, and 100% and 81% in the LSIL group, respectively. Two observers had a high concordance in assessing p16^INK4a stained cells (κ value of 0.841).

CONCLUSIONS.

These data suggested that the use of p16^INK4a as a biomarker combined with nuclear scoring of p16^INK4a positive cells in cervical cytology to triage ASCUS and/or LSIL cases allows identification of HGCIN with good sensitivity and specificity. Cancer (Cancer Cytopathol) 2007. © 2006 American Cancer Society.     

Impact of the more restrictive definition of atypical squamous cells introduced by the 2001 Bethesda System on the sensitivity and specificity of the Papanicolaou test

BACKGROUND.

The Bethesda 2001 (B2001) classification system raised the threshold for reporting atypical squamous cells. The category of Atypical Squamous Cells of Undetermined Significance (ASCUS)‐favor reactive has been eliminated with a recommendation for cytologists to judiciously downgrade those Papanicolaou (Pap) tests that would formerly have been in this category to Negative for Intraepithelial Lesion or Malignancy (NILM). The effect of this change on sensitivity and specificity of the Pap test is not yet known.

METHODS.

A total of 535 consecutive SurePath Pap tests interpreted as ASCUS during a period from March 1 through December 31, 2001, with follow‐up polymerase chain reaction(PCR)‐based human papilloma virus (HPV) testing, were independently reviewed by 3 pathologists and 1 cytotechnologist who reinterpreted these tests according to Bethesda 2001 criteria. Follow‐up HPV and biopsy results were compiled for a 5‐year period.

RESULTS.

By consensus of the 4 observers, 169 (32%) of the ASCUS cases were downgraded to NILM. These cases showed a lower rate of underlying high‐risk HPV infection (11% vs 30%) and cervical intraepithelial neoplasia of grades 2 to 3 (CIN 2/3) (5% vs 10%) on follow‐up than those tests that were reinterpreted as ASCUS or higher. Nine women with follow‐up CIN 2/3 would have had the Pap test interpreted as NILM instead of ASCUS under Bethesda 2001 (20% of all CIN 2/3 found). Individual reviewers downgraded 29% to 42% Pap tests to NILM including those of 5 to 10 women with follow‐up CIN 2/3.

CONCLUSIONS.

The ASCUS threshold established by B2001 prevents a sizeable subset of women from having follow‐up for ASC. However, as a consequence, a few women with CIN 2/3 are downgraded to NILM. The cost savings thus achieved must be weighed against the lost opportunities to detect CIN 2/3. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Diagnostic value of p16INK4A,Ki‐67, and human papillomavirus L1 capsid protein immunochemical staining on cell blocks from residual liquid‐based gynecologic cytology specimens

BACKGROUND:

This study was conducted to evaluate the reliability and role of cell block preparations in the diagnosis of neoplastic and preneoplastic lesions of the cervix and to improve the value of cell block preparations in diagnosing and predicting the prognosis of cervical lesions through immunostaining of p16INK4A (p16), Ki‐67, and human papillomavirus (HPV) L1 capsid protein (HPV L1).

METHODS:

In total, 138 specimens were diagnosed on liquid‐based cytology (LBC) and cell block preparations, and 63 specimens were subjected subsequently to tissue follow‐up and immunostaining for p16, Ki‐67, and HPV L1 on cell block sections.

RESULTS:

In 42 specimens that were diagnosed as low‐grade squamous intraepithelial lesion, high‐grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) on cell blocks, 38 specimens (90.5%) were confirmed by histopathologic reports, and there was slightly better than 81.6% agreement between LBC and tissue follow‐up. Immunointensity and cells that were positive for p16 were enhanced according to increased pathologic grade and differed statistically between cervical intraepithelial neoplasia 1 (CIN‐1) and CIN‐2/CIN‐3 as well as SCC. The positive rates of HPV L1 decreased gradually according to the severity of cervical neoplasia, and HPV L1/p16 expression patterns were related to the severity of cervical lesions.

CONCLUSIONS:

The cell block preparation technique was complementary to LBC, and the authors concluded that the application of LBC combined with cell block preparations may improve the diagnostic accuracy of cytology. Immunostaining for p16 and Ki‐67 on cell block preparations can help to improve the diagnostic accuracy of HSIL and SCC. A combined expression pattern of p16 and HPV L1 may serve as a valuable index for predicting prognosis and follow‐up of cervical dysplastic lesions. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.     

Use of high-risk human papillomavirus testing in patients with low-grade squamous intraepithelial lesions

BACKGROUND

The role of testing for high‐risk human papillomavirus (HR HPV) when triaging women with a cytologic diagnosis of low‐grade squamous intraepithelial lesion (LSIL) has not been well established. The objective of the current study was to correlate the status of HR HPV with the incidence of cervical intraepithelial neoplasia 2 and more severe lesions (CIN 2+) on tissue follow‐up in women with LSIL.

METHODS

A total of 1046 women with LSIL and HR HPV testing were identified in the database of a large teaching hospital within a 12‐month period. HR HPV testing was performed using the Hybrid Capture 2 assay with 1 relative light unit/cutoff as the cutoff.

RESULTS

Of the 1046 women with LSIL and concurrent HR HPV testing, 82.3% tested positive for HR HPV, 91.1% of whom were women aged < 30 years and 73% of whom were women aged ≥ 30 years (P < .001). Cytologic and/or histologic follow‐up was available in 979 (93.6%) women; 25.5% had negative follow‐up, 62.5% were found to have CIN 1 lesions, and 12.0% had CIN 2+ lesions. The sensitivity and negative predictive value of HR HPV status as a marker of CIN 2+ lesions were 98.3% and 98.9%, respectively. The colposcopy rate was 73.3% and 96.9% for women aged ≥ 30 years and women aged < 30 years, respectively (P = .01).

CONCLUSIONS

Using 1 RLU/CO as the cutoff value, HR HPV testing was found to be highly sensitive for detecting CIN 2+ lesions in women with LSIL. The colposcopy rate was significantly lower in women aged ≥ 30 years compared with women aged < 30 years. Triaging with HR HPV testing may be indicated in women aged ≥ 30 years with LSIL cytology, but not in women aged < 30 years. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Combined use of cytology,p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.     

Accuracy of liquid‐based cytology

BACKGROUND:

In the New Technologies for Cervical Cancer Screening (NTCC) randomized controlled trial, no significant increase in the sensitivity of liquid‐based cytology (LBC) was observed compared with conventional cytology. Both were interpreted by cytologists who had limited previous LBC experience. The objective of the current study was to assess whether different results could be expected with experienced LBC interpreters.

METHODS:

A stratified, random sample of 818 LBC slides from the NTCC study was obtained. These slides were reviewed blindly and independently by 3 international experts who did not participate in the NTCC. The sensitivity and specificity of external experts were estimated for cervical intraepithelial neoplasia grade 2 or greater (CIN2+) and for CIN3+ histology, and the differences were compared with the sensitivity and specificity of the original cytologic interpretation using cutoffs of atypical squamous cells of undetermined significance (ASCUS) and low‐grade squamous intraepithelial lesion (LSIL).

RESULTS:

With the endpoint of CIN2+ histology, the difference in sensitivity between external experts and the original interpretation was ?5.3 (95% confidence interval [CI], ?16.0 to 5.4) with ASCUS as the cutoff and 3.8 (95% CI, ?8.2 to 15.8) with LSIL as the cutoff. External experts had slightly lower specificity using ASCUS as the cutoff (?3.4; 95% CI, ?3.9 to ?2.9) and LSIL as the cutoff (?0.7; 95% CI, ?1.0 to ?0.4).

CONCLUSIONS:

The accuracy of the external experts' interpretation was similar to that of the original interpretation. Therefore, the current results indicated that LBC is not expected to increase sensitivity even if it is used by interpreters who have extensive experience with this technique. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

HPV triage for low grade (L-SIL) cytology is appropriate for women over 35 in mass cervical cancer screening using liquid based cytology

In the experimental arm of a randomised trial, women were tested both for liquid-based cytology and human papillomavirus (HPV) DNA and referred for colposcopy if cytology was ASCUS (atypical cells of undetermined significance) or more severe. We considered those with ASCUS (757) or LSIL (low-grade squamous intraepithelial lesions) (485) and a valid HPV test who received colposcopy. We computed sensitivity, specificity and ROC curves with different values of relative light units (RLU, that are related to viral load) as cut off, using cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) at blind histology review as the endpoint. The area under the receiver operating characteristic (ROC) curve was significantly less among women aged 25-34 years than in those older, both considering ASCUS/AGUS (atypical glandular cells of undetermined significance) (p=0.0355) and LSIL (p=0.0009). At age 35-60 the curves for ASCUS and LSIL were similar, while at age 25-34 the area under the curve for LSIL was significantly less than for ASCUS (p=0.0084). With LSIL cytology, specificity of Hybrid Capture 2 with 2 RLU cut-off was 35.0% (95%CI 28.4-42.1) at age 25-34 and 64.5% (95%CI 58.3-70.3) at age 35-60. In conclusion, triaging by HPV testing performed better in women aged over 35 years than those younger. For older women, HPV triaging should also be considered for managing those with LSIL cytology.     

Combined p16INK4a and human papillomavirus testing improves the prediction of cervical intraepithelial neoplasia (CIN II-III) in Thai patients with low-grade cytological abnormalities

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.     

Hybrid capture 2 human papillomavirus DNA testing for women with atypical squamous cells of undetermined significance papanicolaou results in SurePath and ThinPrep specimens

BACKGROUND:

Human papillomavirus (HPV) DNA testing using Hybrid Capture 2 assay with ThinPrep Papanicolaou (Pap) collection is the only US Food and Drug Administration‐approved method for the triage of women with atypical squamous cells of undetermined significance (ASCUS). Although SurePath Pap collection has been used for Hybrid Capture 2 HPV DNA testing, clinical validation of this method has been scarce.

METHODS:

From a cervical cancer‐screening program in Mississippi, we analyzed data from screenings of 8380 women with ASCUS Pap results who underwent reflex Hybrid Capture 2 HPV DNA tests during a course of 4 years. Of these, 4145 were screened with the ThinPrep collection system, and 4235 were screened with SurePath. Results of follow‐up biopsies within 3 months of Pap tests were available for the ThinPrep group (229 cases) and the SurePath group (455 cases). Hybrid Capture 2 positive rates and the follow‐up biopsy results from both groups were compared.

RESULTS:

Hybrid Capture 2 detected high‐risk HPV DNA in 68.8% of ThinPrep and 66.7% of SurePath‐collected specimens (P = .37). Detection rates for CIN2+ and CIN3+ were also comparable between ThinPrep (21.4%, 3.1%) and SurePath (15.4%, 4.2%) using Hybrid Capture 2 (P = .06, P = .45). In ThinPrep‐collected specimens, 4.4% were quantitatively insufficient for Hybrid Capture 2 testing. Significantly more equivocal Hybrid Capture 2 results were observed in SurePath (11.4%) than in ThinPrep specimens (3.2%). However, 67.4% of women with equivocal Hybrid Capture 2 results had negative 1‐year Pap cytology follow‐up in the SurePath group.

CONCLUSIONS:

Hybrid Capture 2 positive rates and CIN2‐3 detection rates were comparable for the SurePath and ThinPrep Pap collection systems, thus supporting the use of SurePath for Hybrid Capture 2 testing. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Image‐guided ThinPrep Papanicolaou tests and cotesting with high‐risk human papillomavirus in women aged 30 years and older in a low‐risk private practice population

BACKGROUND:

Screening for cervical cancer precursors has evolved considerably with the introduction of new technologies to improve the early detection of disease. The objective of this study was to analyze the accuracy and effectiveness of combined screening with cytology and high‐risk human papillomavirus (HR‐HPV) testing in a low‐risk population of women aged ≥30 years.

METHODS:

Consecutive unselected samples from a group of 1871 women aged ≥30 years were screened with image‐guided ThinPrep tests and HR‐HPV tests during a 6‐month period. Histologic follow‐up was reviewed among women with positive HR‐HPV tests.

RESULTS:

A total of 85 (4.5%) women had positive HR‐HPV tests. In 48 HR‐HPV–positive women with follow‐up biopsies, 41 (85%) were found to have histologic abnormalities. Thirty‐three (1.9%) women with cytologically normal Papanicolaou (Pap) tests harbored HR‐HPV, and a cervical intraepithelial neoplasia (CIN) 2+ lesion was detected in 1 (16%) of 6 women with histologic follow‐up. Conversely, 2 (28%) of 7 women with high‐grade intraepithelial lesion on cytology tested negative for HR‐HPV during the same period. A case of serous carcinoma with atypical glandular cells on cytology was also negative for HR‐HPV, as expected.

CONCLUSIONS:

In this low‐risk population of women aged ≥30 years, histology‐confirmed CIN2+ lesions were identified in women with negative cytology and positive HR‐HPV tests, as well as in those with positive cytology and negative HR‐HPV tests. Because both cytology and HPV testing alone missed significant lesions, cotesting with Pap and HR‐HPV in women aged ≥30 years appears to be a reasonable option in a low‐risk population. (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Invader human papillomavirus (HPV) type 16 and 18 assays as adjuncts to HPV screening of cervical papanicolaou smears with atypical squamous cells of undetermined significance

BACKGROUND:

High‐risk (HR) human papillomavirus (HPV) testing is standard practice for triaging women who have Papanicolaou (Pap) smears with atypical squamous cells of undetermined significance (ASC‐US), however, only 5% to 17% of these women have underlying cervical intraepithelial neoplasia 2 (CIN‐2)/CIN‐3. Recent reports have demonstrated that the presence of either HPV type 16 (HPV‐16) or HPV‐18 confers an elevated risk for CIN‐2/CIN‐3. The current study was designed to determine the prevalence of HPV‐16 and HPV‐18 in ASC‐US Pap smears and to determine whether further typing would enhance the risk stratification of patients for CIN‐2/CIN‐3.

METHODS:

One hundred seventy‐eight Pap smears with ASC‐US were screened retrospectively for HR HPV by using the proprietary Invader screening assay followed by typing for HPV‐16 and HPV‐18 by using Invader type‐specific probes on 100 of the samples. Clinical follow‐up results were correlated with HPV types.

RESULTS:

Fifty‐one percent of the ASC‐US samples were positive for HR HPV, the majority of which (70%) harbored non‐HPV‐16/HPV‐18 HR HPV types; 27% were associated with HPV‐16, whereas only 3% contained HPV‐18. The screening assay indicated that 46% of women who had Pap smears with ASC‐US were in need of further HPV‐16/HPV‐18 typing. Testing for HPV‐16 stratified women with ASC‐US into 3 groups: 1) 14% of women were positive for HPV‐16 and had a high risk (54%) of CIN‐2/CIN‐3 on follow‐up biopsy, 2) 35% of women were positive for non‐HPV‐16 HPV types and had an intermediate risk (9%), and 3) 51% of women were negative for HPV and had a negligible risk for CIN‐2/CIN‐3.

CONCLUSIONS:

The combined application of a proprietary screening assay and a type‐specific HPV‐16 assay demonstrated global potential for the development of tailored management protocols for women who have Pap smears with ASC‐US. Cancer 2009. © 2009 American Cancer Society.     

Does the interval between papanicolaou tests influence the quality of cytology?

BACKGROUND: It is commonly believed that the sensitivity of Papanicolaou (Pap) tests decreases with a short interval between cytology samplings. To the authors' knowledge, there is only limited evidence to support this belief. METHODS: For 5055 women in the Atypical Squamous Cells of Undetermined Significance (ASCUS)-Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS), the Pap interval was defined as the number of days between the referral Pap smear demonstrating ASCUS or LSIL ("first cytology") and the enrollment liquid-based ("repeat") cytology. The authors investigated the influence of the interval between Pap smears on repeat cytology by examining percentages of abnormal findings, cellularity, and test sensitivity among women diagnosed with histologic grade 3 cervical intraepithelial neoplasia (CIN3) during the 2-year course of the ALTS. In addition, because human papillomavirus (HPV) DNA adjunct testing is now performed, the authors evaluated HPV viral load, which was assayed using residual liquid cytology specimens, in women with CIN3. RESULTS: The Pap interval ranged from 8-30 days in 763 women, 31-60 days in 2317 women, 61-90 days in 1090 women, 91-120 days in 491 women, and 121-184 days in 394 women (mean of 61.3 days; standard deviation of 34 days). Repeat cytologic interpretations of unsatisfactory findings, ASCUS, and high-grade squamous intraepithelial lesion (HSIL) did not appear to vary among the Pap interval groups. However, low-grade cytologic regression occurred with an increasing Pap interval; negative cytology increased from 28.3% (8-30 days) to 41.6% (121-184 days) (P < 0.0001) whereas LSIL cytology decreased (P trend = 0.002). The approximate cellularity of the samples was slightly better in the interval group of 8-30 days (P trend = 0.04). Among women with CIN3, the repeat test sensitivity at a threshold of ASCUS or greater and the HPV DNA viral load was not found to vary by Pap interval (P trend = 0.80 and P trend = 0.36, respectively). CONCLUSIONS: The authors concluded that a short Pap interval (range, 15-120 days) does not significantly affect the quality of liquid-based repeat cytology, nor the viral load tested from a residual liquid-based specimen.     

P16/Ki-67 Dual Staining in Positive Human Papillomavirus DNA Testing for Predictive Diagnosis of Abnormal Cervical Lesions in Northeastern Thai Women

Objective: Cervical cancer screening can effectively reduce new cervical cancer cases, including in Thailand. The abnormal results are subsequently referred for colposcopy. To avoid unnecessary colposcopy, an efficient triage is still needed for validation. This study aimed to investigate the overall positivity of cytology-based screening, HPV detection, and p16/Ki-67 dual staining and evaluate different triage strategies for predictive diagnosis of abnormal cervical lesions in northeastern Thailand. Methods: Cervical cells were collected from 191 women who came for cervical screening in the gynecological outpatient department during March 2019-February 2020. Pap smear samples were classified into 6 groups including 17 atypical glandular cells (AGC), 21 atypical squamous cells of undetermined significance (ASC-US), 7 atypical squamous cells - cannot exclude HSIL (ASC-H), 26 low-grade squamous intraepithelial lesions (LSILs), 19 high-grade SILs (HSILs) and 101 no squamous intraepithelial lesion (noSIL). Polymerase chain reaction (PCR) was performed for HPV DNA detection. HPV genotyping was determined by reverse line blot hybridization. P16/Ki-67 dual staining was performed by using CINtec PLUS Cytology kit. Biopsies from abnormal screening were collected for surgical pathology classification. Results: High-risk HPV (HR-HPV) infection was 2.97%, 29.41%, 38.10%, 57.14%, 46.15% and 84.21% in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL cytology respectively. P16/ Ki-67 in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL was 0.99%, 5.88%, 9.52%, 42.86%, 26.92% and 63.16%, respectively (P-value < 0.001). Among p16/Ki-67 positive cases, 96.15% (25/26) were infected with HPV and 84.62% (22/26) were HR-HPV. The overall positivity of each and co-testing between cytology or HPV DNA testing or p16/Ki-67 dual staining was evaluated. In each cervical lesion, primary HPV DNA testing showed the highest sensitivity, but low specificity. The combined all HPV/HR-HPV with p16/Ki-67 detection increased the specificity of abnormal cervical lesions. Conclusion: P16/Ki-67 dual stain cytology in HPV-positive women performs well for diagnosis of abnormal cervical lesions and should be considered for management of HPV-positive women to avoid unnecessary colposcopy referrals.