Clinical Review

Atypical squamous cells: The case for HPV testing

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New data, terminology, and guidelines are in, and human papillomavirus testing is emerging as the most efficient and cost-effective triage option.



  • Management by immediate colposcopy, repeat cytology, or HPV testing is acceptable for ASC-US, but testing for HPV is preferred when the Pap test is liquid-based.
  • The sensitivity of HPV triage for high-grade CIN is essentially equivalent to colposcopy, and reduces the need for colposcopy by half.
  • HPV testing is a good option for follow-up after treatment with cryosurgery, loop electrosurgical excision procedure, laser, or cold-knife conization.

What’s the best management strategy for the roughly 2 to 3 million women each yearUpdate on Cervical Disease, for commentary by Thomas C. Wright, Jr, MD, Department of Pathology, College of Physicians and Surgeons of Columbia University.The difference is even more pronounced when the cumulative 2-year detection rate for CIN 2,3 is added in for women referred for HPV-positive ASCUS but not found to have CIN 2,3 at initial colposcopy. That rate rises from 20.1% at initial colposcopy to 26.9% at 2 years.8 Although many experts consider even HPV-positive ASCUS of minimal risk, few would consider a risk of high-grade disease exceeding 1 in 4 to be minimal. In fact, 39% of the total CIN 2,3 cases reported from a routine screening population were detected following triage of ASCUS, and fully 69% were from all equivocal and low-grade Pap diagnoses.9


Risk of cervical intraepithelial neoplasia grade 2 or greater at initial colposcopy

Cox6Hybrid capture 1 (expanded first–generation test)17% (14/81)0.74% (1/136)6.9% (15/217)
Manos7Hybrid capture 215% (45/300)1.2% (6/498)6.4% (51/798)
Solomon4 (ALTS)Hybrid capture 218% (195/1,087)1.1% (13/1,175)9.2% (208/2,262)
ALTS = ASCUS/LSIL Triage Study; ASCUS = atypical squamous cells of undetermined significance; HPV = human papillomavirus

Bethesda 3 redefines ASCUS

The third Bethesda System workshop took place in May 2001 with the aim of evaluating and updating earlier terminology.10 It began by eliminating the words “of undetermined significance” from the overall ASCUS category, which is now called simply “atypical squamous cells,” or ASC. Most subcategories of the former ASCUS were eliminated as well. (Note: Within this article, the acronyms ASCUS and ASC-US are both used to describe atypical squamous cells of undetermined significance. The latter acronym reflects usage and guidelines developed after the third Bethesda workshop.)

Now the ASC classification is broken down into 2 distinct groups:

Atypical squamous cells–undetermined significance, or ASC-US. This new subcategory includes cells previously termed “favor reactive” but not relegated by the pathologist to normal, as well as cells previously in the “unqualified” and “favor HPV” or “favor low-grade squamous intraepithelial lesion (LSIL)” subcategories.

Atypical squamous cells–cannot rule out high-grade squamous intraepithelial lesions, or ASC-H. This category includes atypical cells difficult to distinguish from high-grade cells but not definitive for that classification. Women with such Pap tests are at greater risk for high-risk HPV and histologic CIN 2,3 (TABLE 2).

Evidence-based guidelines reflect Bethesda 3 changes. By the time of Bethesda 3, extensive new data on the management of abnormal cytology was available, including but not limited to data from ALTS, making it possible to create evidence-based guidelines on management of abnormal cervical cytology and CIN. These guidelines were developed in 2001 at a consensus conference hosted by the American Society for Colposcopy and Cervical Pathology (ASCCP),11 with input from 29 professional organizations, federal agencies, and national and international health organizations.

The entire set recommendations for all types of abnormal Pap tests were published in the April 24, 2002 issue of the Journal of the American Medical Association, and management recommendations for histologically proven CIN were published in the July 2003 American Journal of Obstetrics and Gynecology and the July 2003 Journal of Lower Genital Tract Disease. The management algorithms for both cytology and histology can be downloaded from


Comparison of risk for high-risk HPV and CIN grade 2,3, by Pap results

Data from Sherman et al29
ASC-US = atypical squamous cells–undetermined significance;
ASC-H = Atypical squamous cells–cannot rule out high-grade squamous intraepithelial lesion; CIN = cervical intraepithelial neoplasia; HPV = human papillomavirus; HSIL = high-grade squamous intraepithelial lesion

All 3 triage options safe, effective

An evidence-based review found all 3 options safe and effective.11 Therefore, management by immediate colposcopy, repeat cytology, or HPV testing is acceptable for ASCUS, but testing for HPV is preferred when the Pap test is liquid-based (FIGURE 1).

Liquid-based cytology (ThinPrep; Cytyc, Boxborough, Mass and SurePath, Raleigh-Durham, NC) has several advantages. For example, residual cells in the fluid can be tested for HPV, eliminating a return visit.

Immediate colposcopy: Low predictive value, high anxiety and expense. Proponents of immediate colposcopy for all women with ASC-US argue that this would theoretically detect all CIN 2,3 and cancer. However, the positive predictive value of this approach will always be extremely low due to the low rate (6.4% to 11.9%) of CIN 2,3 in women with ASCUS.4,6,7 What’s more, the cost and anxiety generated by immediate colposcopy are high.12


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