p16INK4a Cytology vs HPV Testing for Detecting High-Grade Cervical Disease
p16INK4a Cytology vs HPV Testing for Detecting High-Grade Cervical Disease
We analyzed the performance of p16 immunocytochemistry on a series of 810 retrospectively collected atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) cases with available biopsy follow-up data, including 94 cases of cervical intraepithelial neoplasia (CIN) 2 and 128 cases of CIN 3. Human papillomavirus (HPV) testing was performed from the same residual liquid-based cytologic specimen, and results for both tests were correlated with histologic follow-up data. Sensitivity values for high-grade CIN (HGCIN) confirmed on biopsy within 6 months were 92.6% (ASC-US) and 92.2% (LSIL) for cytotechnologists' reviews of p16 cytology and 90.1% (ASC-US) and 95.7% (LSIL) for HPV testing. Sensitivity rates of initial pathologists' reviews were slightly lower, 76.4% to 80.1%, with levels comparable to cytotechnologists' results after adjudication. The specificity of p16 cytology for HGCIN detection was significantly higher than for HPV testing for cytotechnologists and pathologists: 63.2% to 71.1% (p16 cytology) vs 37.8% for HPV in ASC-US (P < .001) and 37.3% to 53.3% (p16 cytology) vs 18.5% for HPV in LSIL (P < .001). This evaluation of the diagnostic performance of p16 cytology confirms the potential of this stain for the efficient triage of ASC-US and LSIL cytologic results.
Owing to the low prevalence of high-grade precancerous cervical lesions in the routine screening population, there is a substantial number of Papanicolaou (Pap) cytologic cases with equivocal (atypical squamous cells of undetermined significance [ASC-US]) or mildly abnormal (low-grade squamous intraepithelial lesion [LSIL]) morphologic findings for which no high-grade cervical intraepithelial neoplasia (HGCIN) can be confirmed on histologic specimens collected during subsequent colposcopy-guided biopsy sampling (reviewed by Schiffman et al and Wright et al). Although to a substantial extent colposcopic error is known to lead to failure in finding HGCIN on first referral to colposcopy, there is a large proportion of women within these 2 Pap cytologic categories who neither have nor may develop HGCIN over time. Thus, it is important to implement an efficient triage approach for ASC-US and LSIL Pap cytologic results to identify women at highest risk for underlying precancerous disease and who require immediate further diagnostic follow-up.
Because most cervical cancers are induced by persistent infection with high-risk human papillomavirus (HR-HPV) types, testing for the presence of HPV has been incorporated into the management of Pap cytologic results categorized as ASC-US in various countries. The ASC-US–LSIL Triage Study (ALTS) has shown that women with an ASC-US result and being negative for HR-HPV have a very low risk of having an HGCIN. In that study, the triage of ASC-US cases with HPV testing has been found to be similarly effective as repeated cytology or direct colposcopy, with a slightly favorable trade-off of sensitivity and referral rates of HPV triage over repeated Pap cytology.
However, despite a high sensitivity for the detection of underlying HGCIN within the ASC-US group, testing for HPV has limitations, especially in terms of specificity. The vast majority of ASC-US cases testing positive for HR-HPV have no underlying high-grade abnormality. Furthermore, given the high prevalence of HPV infections, especially in younger age groups, the effectiveness of triaging ASC-US cases with HPV testing is variable, depending on the patient's age and other socioeconomic factors.
For the management of cytologic results categorized as LSIL, there is currently no triage tool available that could assist in determining which women with such low-grade cytologic abnormalities should undergo colposcopy, and, therefore, colposcopy is standard for most patients. The ASC-US–LSIL Triage Study and other studies have shown that cytologic interpretations of LSIL are so widely associated with infections with HR-HPV types that there is no clinical usefulness for triaging LSIL cytologic results with testing for the presence of HPV infections.
The relatively low threshold of an ASC-US result positively triaged with HPV testing or an LSIL Pap cytologic result triggering referral to colposcopy greatly enhances sensitivity for the detection of HGCIN, but at the cost of referring most of the women to colposcopy and biopsy follow-up who do not have prevalent CIN 2 or CIN 3 disease or invasive cervical cancer. Therefore, the availability of a marker that provides a sensitivity similar to that of HPV testing but with a significantly higher specificity would be desirable to improve current triage testing of ASC-US cytologic results and to allow for reducing the colposcopy referral rates after LSIL on cytology.
In recent years, various molecular markers have been proposed as potential candidates for the efficient triage of equivocal and mildly abnormal Pap cytologic results (reviewed by Cuschieri and Wentzensen and Wentzensen and von Knebel Doeberitz). One of the most intensely studied markers is the cellular p16 protein (p16), a cyclin-dependent kinase inhibitor that has been demonstrated in a large number of studies to be strongly overexpressed in almost all high-grade precursor lesions and invasive cancers of the cervix uteri (reviewed by Cuschieri and Wentzensen, Wentzensen and von Knebel Doeberitz, and Tsoumpou et al). The expression of this cell cycle regulatory protein has been shown to be directly linked to the transforming activity of the viral E7 oncoprotein at the molecular level. As the continuous inactivation of the retinoblastoma protein (pRb) by the viral E7 oncoprotein is necessary to maintain the malignant phenotype of HPV-associated cancer cells, detection of overexpression of p16 may be used as a highly sensitive and specific surrogate marker for the inactivation of the tumor suppressor pRb in HPV-transformed epithelial cells (reviewed by Wentzensen and von Knebel Doeberitz).
The usefulness of conjunctive testing for p16 overexpression in cervical tissue specimens has been shown in many studies and is widely accepted (reviewed by Cuschieri and Wentzensen and Tsoumpou et al). In cervical cytology, various research studies have been performed to evaluate the potential usefulness of applying p16 immunocytochemical staining protocols, especially for the triage of equivocal or mildly abnormal Pap cytologic results. In most studies, a sensitivity similar to that of HPV testing, but at a substantially higher specificity rate, has been reported for p16 cytology when used for the triage of Pap cytologic results categorized as ASC-US or LSIL or containing atypical glandular cells (reviewed by Cuschieri and Wentzensen and Tsoumpou et al). Although this indicates the potential of p16 cytologic testing for the triage of abnormal Pap cytologic results, many of the research studies show limitations owing to the use of non-validated and nonstandardized immunocytochemical reagents and protocols, the lack of standardized interpretation protocols, and a low number of biopsy follow-up results available.
We therefore set out to perform a retrospective, multi-center, multinational, diagnostic case control study to validate the performance characteristics of the CINtec p16 Cytology test (mtm laboratories, Heidelberg, Germany) on a large number (n = 810) of residual liquid-based cervical cytologic specimens with follow-up biopsy specimens available.
Abstract and Introduction
Abstract
We analyzed the performance of p16 immunocytochemistry on a series of 810 retrospectively collected atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) cases with available biopsy follow-up data, including 94 cases of cervical intraepithelial neoplasia (CIN) 2 and 128 cases of CIN 3. Human papillomavirus (HPV) testing was performed from the same residual liquid-based cytologic specimen, and results for both tests were correlated with histologic follow-up data. Sensitivity values for high-grade CIN (HGCIN) confirmed on biopsy within 6 months were 92.6% (ASC-US) and 92.2% (LSIL) for cytotechnologists' reviews of p16 cytology and 90.1% (ASC-US) and 95.7% (LSIL) for HPV testing. Sensitivity rates of initial pathologists' reviews were slightly lower, 76.4% to 80.1%, with levels comparable to cytotechnologists' results after adjudication. The specificity of p16 cytology for HGCIN detection was significantly higher than for HPV testing for cytotechnologists and pathologists: 63.2% to 71.1% (p16 cytology) vs 37.8% for HPV in ASC-US (P < .001) and 37.3% to 53.3% (p16 cytology) vs 18.5% for HPV in LSIL (P < .001). This evaluation of the diagnostic performance of p16 cytology confirms the potential of this stain for the efficient triage of ASC-US and LSIL cytologic results.
Introduction
Owing to the low prevalence of high-grade precancerous cervical lesions in the routine screening population, there is a substantial number of Papanicolaou (Pap) cytologic cases with equivocal (atypical squamous cells of undetermined significance [ASC-US]) or mildly abnormal (low-grade squamous intraepithelial lesion [LSIL]) morphologic findings for which no high-grade cervical intraepithelial neoplasia (HGCIN) can be confirmed on histologic specimens collected during subsequent colposcopy-guided biopsy sampling (reviewed by Schiffman et al and Wright et al). Although to a substantial extent colposcopic error is known to lead to failure in finding HGCIN on first referral to colposcopy, there is a large proportion of women within these 2 Pap cytologic categories who neither have nor may develop HGCIN over time. Thus, it is important to implement an efficient triage approach for ASC-US and LSIL Pap cytologic results to identify women at highest risk for underlying precancerous disease and who require immediate further diagnostic follow-up.
Because most cervical cancers are induced by persistent infection with high-risk human papillomavirus (HR-HPV) types, testing for the presence of HPV has been incorporated into the management of Pap cytologic results categorized as ASC-US in various countries. The ASC-US–LSIL Triage Study (ALTS) has shown that women with an ASC-US result and being negative for HR-HPV have a very low risk of having an HGCIN. In that study, the triage of ASC-US cases with HPV testing has been found to be similarly effective as repeated cytology or direct colposcopy, with a slightly favorable trade-off of sensitivity and referral rates of HPV triage over repeated Pap cytology.
However, despite a high sensitivity for the detection of underlying HGCIN within the ASC-US group, testing for HPV has limitations, especially in terms of specificity. The vast majority of ASC-US cases testing positive for HR-HPV have no underlying high-grade abnormality. Furthermore, given the high prevalence of HPV infections, especially in younger age groups, the effectiveness of triaging ASC-US cases with HPV testing is variable, depending on the patient's age and other socioeconomic factors.
For the management of cytologic results categorized as LSIL, there is currently no triage tool available that could assist in determining which women with such low-grade cytologic abnormalities should undergo colposcopy, and, therefore, colposcopy is standard for most patients. The ASC-US–LSIL Triage Study and other studies have shown that cytologic interpretations of LSIL are so widely associated with infections with HR-HPV types that there is no clinical usefulness for triaging LSIL cytologic results with testing for the presence of HPV infections.
The relatively low threshold of an ASC-US result positively triaged with HPV testing or an LSIL Pap cytologic result triggering referral to colposcopy greatly enhances sensitivity for the detection of HGCIN, but at the cost of referring most of the women to colposcopy and biopsy follow-up who do not have prevalent CIN 2 or CIN 3 disease or invasive cervical cancer. Therefore, the availability of a marker that provides a sensitivity similar to that of HPV testing but with a significantly higher specificity would be desirable to improve current triage testing of ASC-US cytologic results and to allow for reducing the colposcopy referral rates after LSIL on cytology.
In recent years, various molecular markers have been proposed as potential candidates for the efficient triage of equivocal and mildly abnormal Pap cytologic results (reviewed by Cuschieri and Wentzensen and Wentzensen and von Knebel Doeberitz). One of the most intensely studied markers is the cellular p16 protein (p16), a cyclin-dependent kinase inhibitor that has been demonstrated in a large number of studies to be strongly overexpressed in almost all high-grade precursor lesions and invasive cancers of the cervix uteri (reviewed by Cuschieri and Wentzensen, Wentzensen and von Knebel Doeberitz, and Tsoumpou et al). The expression of this cell cycle regulatory protein has been shown to be directly linked to the transforming activity of the viral E7 oncoprotein at the molecular level. As the continuous inactivation of the retinoblastoma protein (pRb) by the viral E7 oncoprotein is necessary to maintain the malignant phenotype of HPV-associated cancer cells, detection of overexpression of p16 may be used as a highly sensitive and specific surrogate marker for the inactivation of the tumor suppressor pRb in HPV-transformed epithelial cells (reviewed by Wentzensen and von Knebel Doeberitz).
The usefulness of conjunctive testing for p16 overexpression in cervical tissue specimens has been shown in many studies and is widely accepted (reviewed by Cuschieri and Wentzensen and Tsoumpou et al). In cervical cytology, various research studies have been performed to evaluate the potential usefulness of applying p16 immunocytochemical staining protocols, especially for the triage of equivocal or mildly abnormal Pap cytologic results. In most studies, a sensitivity similar to that of HPV testing, but at a substantially higher specificity rate, has been reported for p16 cytology when used for the triage of Pap cytologic results categorized as ASC-US or LSIL or containing atypical glandular cells (reviewed by Cuschieri and Wentzensen and Tsoumpou et al). Although this indicates the potential of p16 cytologic testing for the triage of abnormal Pap cytologic results, many of the research studies show limitations owing to the use of non-validated and nonstandardized immunocytochemical reagents and protocols, the lack of standardized interpretation protocols, and a low number of biopsy follow-up results available.
We therefore set out to perform a retrospective, multi-center, multinational, diagnostic case control study to validate the performance characteristics of the CINtec p16 Cytology test (mtm laboratories, Heidelberg, Germany) on a large number (n = 810) of residual liquid-based cervical cytologic specimens with follow-up biopsy specimens available.
