Abnormal Cervical Cancer Screening Test and Cancer Precursors

Publication Date: April 1, 2020
Last Updated: September 25, 2022

Recommendations

Surveillance

When patients have an estimated 5-year CIN 3+ risk of less than 0.15% based on past history and current test results, return to routine screening at 5-year intervals using HPV-based testing is recommended. (A, II)
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When patients have an estimated 5-year CIN 3+ risk of 0.15% or greater but less than 0.55% based on history and current test results, repeat testing in 3 years with HPV-based testing is recommended. (A, II)
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When patients have an estimated risk of CIN 3+ based on history and current results that is below the threshold for immediate colposcopy (4.0% immediate risk) and above the 3-year follow-up threshold (≥0.55% at 5 years), repeat testing in 1 year with HPV-based testing is recommended. (A, II)
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Colposcopy

When patients have an estimated immediate risk of diagnosis of CIN 3+ of 4.0% or greater based on history and current results, referral to colposcopy is recommended. (A, II)
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For nonpregnant patients 25 years or older with an estimated immediate risk of CIN 3+ of 60% or greater based on history and current results, treatment using an excisional procedure without previous biopsy confirmation is preferred but colposcopy with biopsy is acceptable. (B, II)
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For nonpregnant patients 25 years or older with an estimated immediate risk of CIN 3+ 25% or greater and less than 60% based on history and current results, treatment using an excisional procedure without previous biopsy confirmation or histologic evaluation with colposcopy and biopsy are both acceptable. (A, II)
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2-Tier Terminology

It is important to use p16 immunohistochemical staining according to the guidance provided by the CAP-ASCCP LAST Project. p16 immunohistochemistry should be used for specific indications as recommended by the LAST guidelines when interpreting the hematoxylin and eosin (H&E) slide. A positive p16 immunostain supports the diagnosis of histologic HSIL if the morphological assessment of H&E slides is consistent with CIN 2 or CIN 3. There is a risk of overcalling cervical histology results when p16 is used incorrectly. Most importantly, a morphologic CIN 1 on H&E should not be upgraded to histologic HSIL (CIN 2) even if p16 positive. (, )
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Primary HPV Screening

When primary HPV screening is used, performance of an additional reflex triage test (e.g., reflex cytology) for all positive HPV tests regardless of genotype is preferred (this includes tests positive for genotypes HPV 16/18). (C, III)
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However, if primary HPV screening test genotyping results are HPV 16 or HPV 18 positive and reflex triage testing from the same laboratory specimen is not feasible, referral for colposcopy before obtaining additional testing is acceptable. (C, III)
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If genotyping for HPV 16 or HPV 18 is positive, and triage testing is not performed before the colposcopy, collection of an additional triage test (e.g., cytology) at the colposcopy visit is recommended. (C, III)
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HPV Tests Used in Management

HPV assays should be used for management according to their regulatory approval for screening, unless there are sufficient data to support use of the assay differently. (A, I)
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Evaluation of Cytology Interpreted as AGC or AIS

For nonpregnant patients of all ages with all subcategories of AGC and AIS, except when atypical endometrial cells are specified, colposcopy is recommended regardless of HPV test result; endocervical sampling is recommended at initial colposcopy except in pregnancy. (A, II)
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Accordingly, triage by reflex HPV testing is not recommended, and triage by repeat cytology is unacceptable. (D, II)
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Endometrial sampling is recommended in conjunction with colposcopy and endocervical sampling in nonpregnant patients 35 years or older with all categories of AGC and AIS. (A, II)
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Endometrial sampling is also recommended for nonpregnant patients younger than 35 years at increased risk of endometrial neoplasia based on clinical indications (e.g., abnormal uterine bleeding, conditions suggesting chronic anovulation, or obesity). (A, II)
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For patients with atypical endometrial cells specified, initial evaluation limited to endometrial and endocervical sampling is preferred, with colposcopy acceptable at the time of initial evaluation. If colposcopy was deferred and no endometrial pathology is identified, additional evaluation with colposcopy is then recommended.

Subsequent Management

For patients with cytology showing AGC not otherwise specified or atypical endocervical cells not otherwise specified in whom histologic HSIL (CIN 2+) or AIS/cancer is not identified, cotesting at 1 and 2 years is recommended. If both cotests are negative, repeat cotesting at 3 years is recommended. If any test is abnormal, then colposcopy is recommended. (B, II)
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If CIN 2 or CIN 3 but no glandular lesion is identified histologically for patients with cytology atypical glandular, endocervical, or endometrial cells not otherwise specified, management should be according to the 2019 guidelines for the lesion diagnosed. (C, II)
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For patients with atypical glandular or endocervical cells “favor neoplasia” or endocervical AIS cytology, if invasive disease is not identified during initial colposcopic workup, a diagnostic excisional procedure is recommended. The diagnostic excisional procedure used in this setting should provide an intact specimen with interpretable margins. (B, II)
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Endocervical sampling above the excisional bed is preferred. (B, II)
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Unsatisfactory Cytology

For patients with an unsatisfactory cytology result and no, unknown, or a negative HPV test result, repeat age-based screening (cytology, cotest, or primary HPV test) in 2 to 4 months is recommended. (B, III)
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Triage using HPV testing is not recommended. (D, III)
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Before repeat cytology, treatment to resolve atrophy or obscuring inflammation when a specific infection is present is acceptable. (C, III)
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For patients 25 years and older who are cotested and have unsatisfactory cytology and a positive HPV test without genotyping, repeat cytology in 2 to 4 months or colposcopy is acceptable. (B, II)
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If a positive HPV test with partial genotyping is positive for HPV 16 or HPV 18, direct referral for colposcopy is recommended. (B, II)
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Absent Transformation Zone on Screening Cytology

For patients aged 21 to 29 years with negative screening cytology and absent endocervical cells/transformation zone component (i.e., endocervical cells or squamous metaplastic cells), routine screening is recommended. (B, III)
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When cervical cytology alone is performed for screening, HPV testing as a triage test after negative cytology and absent endocervical cells/transformation zone component in this age group is unacceptable. (D, III)
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For patients 30 years or older with NILM cytology and absent endocervical cells/transformation zone component and no or unknown HPV test result, HPV testing is preferred. (B, III)
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Repeat cytology in 3 years is acceptable if HPV testing is not performed. (B, III)
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Benign Endometrial Cells in Premenopausal Patients or Benign Glandular Cells in Posthysterectomy Patients

For asymptomatic premenopausal patients with benign endometrial cells, endometrial stromal cells, or histiocytes, no further evaluation is recommended. (B, II)
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For postmenopausal patients with benign endometrial cells, endometrial assessment is recommended. (B, II)
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For posthysterectomy patients with a cytology report of benign glandular cells, no further evaluation is recommended. (B, II)
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Exceptions to Colposcopy Threshold

For patients with ASC-H cytology, colposcopy is recommended regardless of HPV result. (A, III)
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For patients with HPV 18–positive NILM, colposcopy is recommended. (A, II)
(Note colposcopy is also recommended for HPV 16–positive NILM, repeated here for clarity.)
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Colposcopy should be performed after 2 consecutive unsatisfactory screening tests. (C, III)
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MANAGING HISTOLOGY RESULTS

Patients 25 Years or Older

Management of Histologic HSIL, not Further Specified or Qualified
Treatment is preferred if histologic HSIL cannot be specified (e.g., reported as histologic HSIL or histologic HSIL [CIN 2,3]). (C, III)
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Management of Histologic HSIL (CIN 2 or CIN 3)
In all nonpregnant patients with a diagnosis of histologic HSIL (CIN 3), treatment is recommended and observation is unacceptable. (A, II)
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In nonpregnant patients with histologic HSIL (CIN 2), treatment is recommended, unless the patient's concerns about the effect of treatment on future pregnancy outweigh concerns about cancer. (B, II)
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Observation is unacceptable when the squamocolumnar junction or the upper limit of the lesion is not fully visualized or when the results of an endocervical sampling, if performed, is CIN 2+ or ungraded. (E, III)
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When treatment of histologic HSIL is planned, excisional treatment is preferred, and treatment with ablation is acceptable. (B, I)
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Outside of the setting of a clinical research trial, nonsurgical therapies, including topical agents, therapeutic vaccines, and other biologics, are unacceptable for the treatment of histologic HSIL (CIN 2 or CIN 3). (D, III)
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Hysterectomy is unacceptable as primary therapy solely for the treatment of histologic HSIL (CIN 2, CIN 3, or unqualified). (E, II)
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When considering ablative therapy, in particular cryotherapy, ablation is unacceptable in the following circumstances. as defined by the WHO:
(a) the lesion extends into the canal and
(b) when the lesion covers more than 75% of the surface area of the ectocervix or extends beyond the cryotip being used.
Additional situations for which cryotherapy is not recommended include the following:
(a) the squamocolumnar junction or the upper limit of any lesion is not fully visualized;
(b) endocervical canal sample is diagnosed as CIN 2+ or CIN that cannot be graded;
(c) after previous treatment for CIN 2+;
(d) in the setting of inadequate biopsies of the cervix to confirm histologic diagnosis; and (e) if cancer is suspected.
(E, III)
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Management of CIN 2 in Those Who Are Concerned About the Potential Effect of Treatment on Future Pregnancy Outcomes
For patients with a diagnosis of histologic HSIL (CIN 2) whose concerns about the effects of treatment on a future pregnancy outweigh their concerns about cancer, either observation or treatment is acceptable provided the squamocolumnar junction is visible and CIN 2+ or ungraded CIN is not identified on endocervical sampling. (C, II)
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If the histologic HSIL cannot be specified as CIN 2, treatment is preferred, but observation is acceptable. (C, III)
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For patients 25 years or older, observation includes colposcopy and HPV-based testing at 6-month intervals for up to 2 years. If during surveillance, all evaluations demonstrate less than CIN 2 and less than ASC-H on 2 successive occasions, 6 months apart, subsequent surveillance should occur at 1 year after the second evaluation and use HPV-based testing. If negative on 3 consecutive annual surveillance tests, proceed to long-term surveillance. If CIN 2 remains present for a 2-year period, treatment is recommended (C, II)
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Management of LSIL (CIN 1) or Less Preceded by ASC-H or HSIL Cytology
When CIN 2+ is not identified histologically after an ASC-H or HSIL cytology result, it is acceptable to review the cytologic, histologic, and colposcopic findings. If the review yields a revised interpretation, management should follow guidelines for the revised diagnosis. (C, III)
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When CIN 2+ is not identified, HSIL cytology is managed more aggressively than ASC-H cytology. For cytology showing HSIL, but biopsy showing histologic LSIL (CIN 1) or less, either an immediate diagnostic excisional procedure or observation with HPV-based testing and colposcopy at 1 year is acceptable, provided in the latter case that the initial colposcopic examination fully visualized the squamocolumnar junction and the upper limit of any lesion, and that the endocervical sampling, if collected, was less than CIN 2. (B, II)
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For ASC-H, if the colposcopic examination can fully visualize the squamocolumnar junction and the upper limit of any lesion and that the endocervical sampling, if collected, is negative, observation at 1 year with HPV-based testing is recommended; a diagnostic excisional procedure is not recommended. (B, II)
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For both HSIL and ASC-H cytology, if observation is elected, and all tests are negative at the 1-year visit, repeat HPV-based testing is recommended in 1 year (at 2 years from the original cytology). If all tests are negative at both the 1- and 2-year follow-up visits, return for retesting with HPV-based testing in 3 years is recommended, then proceed with long-term surveillance. If any test is abnormal during the observation period, repeat colposcopy is recommended, and management based on resulting biopsies is recommended. A diagnostic excisional procedure is recommended for patients with HSIL cytology results at either the 1- or 2-year visit, or ASC-H results that persist at the 2-year visit (C, III)
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Histologic LSIL (CIN 1) Diagnosed Repeatedly for at Least 2 Years
For patients 25 years or older with histologic LSIL (CIN 1) who is diagnosed at consecutive visits for at least 2 years, observation is preferred, (B, II)
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but treatment is acceptable.
(C, III)
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If treatment is selected and the entire squamocolumnar junction and all lesions were fully visualized during colposcopic examination, either excision or ablation treatments are acceptable. (C, II)
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Histologic LSIL (CIN 1) Diagnosed Repeatedly for at Least 2 Years
For patients 25 years or older with histologic LSIL (CIN 1) who is diagnosed at consecutive visits for at least 2 years, observation is preferred (BII) but treatment is acceptable. (C, III)
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If treatment is selected and the entire squamocolumnar junction and all lesions were fully visualized during colposcopic examination, either excision or ablation treatments are acceptable. (C, II)
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Management of AIS
A diagnostic excisional procedure is recommended for all patients with a diagnosis of AIS on cervical biopsy to rule out invasive adenocarcinoma, even when definitive hysterectomy is planned. Excisional procedures should optimally remove an intact specimen to facilitate accurate interpretation of margin status. Although there is no preference for cold knife conization versus LEEP, intentional disruption of the specimen by performance of a LEEP followed by a “top hat” endocervical excision to achieve the desired specimen length is unacceptable. An excisional specimen length of at least 10 mm is preferred, and this can be increased to 18 to 20 mm for patients who are not concerned about the effect of treatment on future pregnancy. These dimensions are preferred regardless of whether hysterectomy is planned.

After the initial diagnostic procedure, hysterectomy is the preferred management for all patients who have a histologic diagnosis of AIS, although fertility-sparing management for appropriately selected patients is acceptable. For patients with confirmed AIS with negative margins on the excisional specimen, simple hysterectomy is preferred. For patients with confirmed AIS with positive margins on the excisional specimen, re-excision to achieve negative margins is preferred, even if hysterectomy is planned. For patients with AIS and persistent positive margins for whom additional excisional procedures are not feasible, either a simple or modified radical hysterectomy is acceptable. After hysterectomy, surveillance per the ASCCP surveillance guidelines for treated CIN 2+ is recommended.

For patients of reproductive age who desire future pregnancy, fertility-sparing management with an excisional procedure is acceptable provided that negative margins have been achieved on the excisional specimen, and the patient is willing and able to adhere to surveillance recommendations. If negative margins cannot be achieved after maximal excisional attempts, fertility-sparing management is not recommended. For patients who undergo fertility-sparing management, surveillance with cotesting and endocervical sampling is recommended every 6 months for at least 3 years, then annually for at least 2 years, or until hysterectomy is performed. For patients who have consistently negative cotesting and endocervical sampling results for 5 years, extending the surveillance interval to every 3 years starting in the sixth year of surveillance is acceptable. Small retrospective studies have shown HPV test results to be the best predictor for recurrent disease. Therefore, for patients who have consistently negative cotesting and endocervical sampling results, continued surveillance is acceptable after completion of childbearing. For patients who have had positive HPV test results or abnormal cytology/histologic results during surveillance, hysterectomy at the completion of childbearing is preferred. (, )
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SURVEILLANCE AFTER ABNORMALITIES

Guidance for Specific Tests and Testing Intervals When Managing Abnormal Results

After abnormal cervical cancer screening test results for patients 25 years or older, colposcopic biopsy results, or treatment of histologic HSIL, surveillance with either HPV testing alone or cotesting is preferred. (A, I)
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Surveillance with cervical cytology alone is acceptable only if testing with HPV or cotesting is not feasible. (C, III)
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Cytology is recommended at 6-month intervals when 1-year intervals are recommended for HPV or cotesting, and annually when 3-year intervals are recommended for HPV or cotesting. (A, II)
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Cytology should be used for patients younger than 25 years, with transition to HPV-based testing at 25 years or older. (A, II)
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Short-Term Follow-up After Treatment for Histologic HSIL

After treatment, HPV-based testing at 6 months is preferred regardless of the margin status of the excisional specimen. (B, II)
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If HPV-based tests are positive, colposcopy and appropriate biopsies should be performed. (A, II)
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Follow-up at 6 months with colposcopy and ECC is acceptable. (B, III)
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Guidance for Long-Term Follow-up After Treatment for High-Grade Histology or Cytology

In patients treated for histologic or cytologic HSIL, after the initial HPV-based test at 6 months, annual HPV or cotesting is preferred until 3 consecutive negative tests have been obtained. (A, II)
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After the initial intensive surveillance period, continued surveillance at 3-year intervals is recommended for at least 25 years after treatment of high-grade histology (histologic HSIL, CIN 2, CIN 3, or AIS) or high-grade cytology (HSIL or persistent ASC-H) even if this is beyond the age of 65 years. (B, II)
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When patients with a history of treated high-grade histology or cytology reach the age of 65 years, if they have completed the initial 25-year surveillance period, continued surveillance at 3-year intervals is acceptable and may continue as long as the patient is in reasonably good health. (B, III)
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Discontinuation of screening is recommended if a patient has a limited life expectancy. Management according to the highest-grade abnormality found on histology or cytology is recommended.

Guidance for Long-Term Follow-up After Low-Grade Cytology (HPV-Positive NILM, ASC-US, or LSIL) or Histologic LSIL (CIN 1) Abnormalities Without Evidence of Histologic or Cytologic High-Grade Abnormalities

Among patients initially diagnosed with low-grade cytology or histologic abnormalities or HPV infections, continued surveillance according to risk estimation using available data is recommended. (C, III)
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SPECIAL POPULATIONS

Management of Patients Younger Than 25 Years

Initial Management After an Abnormal Screening Test Result
In patients younger than 25 years with low-grade cytology screening results of LSIL, ASC-US HPV-positive, or ASC-US without HPV testing, repeat cytology alone at 1 and 2 years after the initial abnormal result is recommended. (B, II)
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Colposcopy is recommended if high-grade cytology is found at any point (HSIL, ASC-H, AGC, AIS) or if low-grade cytology persists at the 2-year follow-up visit. (B, II)
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If reflex HPV testing for ASC-US is performed and the results are negative, repeat cytology in 3 years is recommended. (B, II)
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After 2 consecutive negative cytology results, return to routine age-based screening is recommended. (B, II)
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If colposcopy is performed and the results are less than CIN 2 (i.e., histologic LSIL [CIN 1] or less), repeat cytology in 1 year, Clinicians should switch to using risk estimates when patients reach the age of 25 years. (B, II)
and manage as above (e.g., repeat cytology for ASC-US/LSIL, colposcopy for ASC-H or higher).
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Management of Cytology ASC-H and HSIL in Patients Younger Than 25 Years

Colposcopy is recommended for patients younger than 25 years with ASC-H or HSIL cytology. (A, II)
Immediate treatment without histologic confirmation is not recommended.
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Management of Histology of Less Than CIN 2 Preceded by Cytology ASC-H and HSIL in Patients Younger Than 25 Years

Observation is recommended and diagnostic excisional procedures are not recommended for patients younger than 25 years with a preceding cytology of ASC-H or HSIL and a colposcopy with biopsy of CIN 1 or less as long as the squamocolumnar junction and the upper limit of all lesions are fully visualized, the endocervical sampling is less than CIN 2, and review of histology/cytology does not change the diagnosis. Observation with colposcopy and cytology in 1 and 2 years is recommended for those with HSIL cytology. Cytology at 1 and 2 years is recommended for those with ASC-H cytology, with colposcopy recommended for ASC-US or above on repeat testing. If CIN 2+ is diagnosed, this is managed per guidelines described in the following section. If a high-grade cytologic abnormality (HSIL, ASC-H) without histologic HSIL persists for 2 years, a diagnostic excisional procedure is recommended (unless the patient is pregnant). A diagnostic excisional procedure is recommended in patients when the squamocolumnar junction or the upper limit of all lesions are not fully visualized. (, )
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Management of Histologic HSIL (CIN 2 or CIN 3) for Patients Younger Than 25 Years

In patients younger than 25 years with histologic HSIL (CIN 3), treatment is recommended, and observation is unacceptable. (E, II)
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In patients younger than 25 years with histologic HSIL (CIN 2), observation is preferred, and treatment is acceptable. (B, II)
In patients younger than 25 years with histologic HSIL unspecified as CIN 2 or CIN 3, observation or treatment is acceptable. Observation includes colposcopy and cytology at 6-month intervals. If during surveillance of histologic HSIL, all cytology results are less than ASC-H and histology results are less than CIN 2 at 6 and 12 months, subsequent surveillance should be at 1 year after the second evaluation. If CIN 2 or unspecified histologic HSIL persists for a 2-year period, treatment is recommended. Excisional treatment is recommended when the squamocolumnar junction or the lesion(s) are not fully visualized.
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Managing Patients During Pregnancy

In pregnancy, management of abnormal screening results using the same Clinical Action Thresholds for surveillance and colposcopy established for nonpregnant patients is recommended. (C, III)
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Endocervical curettage, endometrial biopsy, and treatment without biopsy are unacceptable during pregnancy. (E, III)
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A diagnostic excisional procedure or repeat biopsy is recommended only if cancer is suspected based on cytology, colposcopy, or histology. (B, II)
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If histologic HSIL (CIN 2 or CIN 3) is diagnosed at the first colposcopy examination during pregnancy, surveillance colposcopy and testing (diagnostic cytology/HPV depending on age) is preferred every 12 to 24 weeks, but deferring colposcopy to the postpartum period is acceptable. (B, II)
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Repeat biopsy is recommended if invasion is suspected or the appearance of the lesion worsens. (B, II)
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Treatment of histologic HSIL (CIN 2 or CIN 3) during pregnancy is not recommended. (D, II)
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If AIS is diagnosed during pregnancy, referral to a gynecologic oncologist is preferred, but management by a gynecologist skilled in the colposcopic diagnosis and treatment of AIS is acceptable. (C, III)
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In the postpartum period, colposcopy is recommended no earlier than 4 weeks after delivery. (B, II)
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In patients diagnosed with histologic HSIL (CIN2 or CIN3) during pregnancy, if a lesion is detected at postpartum colposcopy, an excisional treatment procedure or full diagnostic evaluation (cervical cytology, HPV, and biopsy) is acceptable. (B, II)
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In the absence of a lesion on colposcopy, a full diagnostic evaluation is recommended; expedited treatment is not recommended. (B, II)
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Managing Patients With Immunosuppression

In immunocompromised patients of any age, colposcopy referral is recommended for all results cytology results of HPV-positive ASC-US or higher. If HPV testing is not performed on ASC-US results, then repeat cytology in 6 to 12 months is recommended, with colposcopy referral for ASC-US or higher. For any result of ASC-US or higher on repeat cytology or if HPV positive, referral to colposcopy is recommended. For all cytology results of LSIL or worse (including ASC-H, AGC, AIS, and HSIL), referral to colposcopy is recommended regardless of HPV test result if done. (, )
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Managing Patients After Hysterectomy

After a diagnosis of high-grade histology or cytology, patients may undergo hysterectomy for reasons related or unrelated to their cervical abnormalities. If hysterectomy is performed for treatment, patients should have 3 consecutive annual HPV-based tests before entering long-term surveillance. Long-term surveillance after treatment for histologic HSIL (CIN 2 or CIN 3) or AIS involves HPV-based testing at 3-year intervals for 25 years, regardless of whether the patient has had a hysterectomy either for treatment or at any point during the surveillance period. (C, III)
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Among patients who have undergone hysterectomy but either have no previous diagnosis of CIN 2+ within the previous 25 years or have completed the 25 year surveillance period, screening is generally not recommended. However, if performed, abnormal vaginal screening test results should be managed according to published recommendations. (B, III)
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Managing Patients Older Than 65 Years With a History of Prior Abnormalities

If patients over age 65 years undergo HPV testing, cotesting, or cytology, management according to guidelines for patients aged 25 to 65 years is recommended. (C, II)
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If surveillance testing is recommended for either a history of abnormal screening results or treatment for precancer, discontinuing surveillance is unacceptable if the patient is in reasonably good health and testing is feasible. (D, II)
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Discontinuation of surveillance is recommended for patients with a limited life expectancy. (E, III)
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Recommendation Grading

Overview

Title

Abnormal Cervical Cancer Screening Test and Cancer Precursors

Authoring Organization

Publication Month/Year

April 1, 2020

Last Updated Month/Year

February 5, 2024

Document Type

Guideline

External Publication Status

Published

Country of Publication

US

Inclusion Criteria

Female, Adult, Older adult

Health Care Settings

Ambulatory, Hospital, Radiology services

Intended Users

Social worker, physician, nurse, nurse practitioner, physician assistant

Scope

Assessment and screening, Diagnosis, Prevention, Management, Treatment

Keywords

Abnormal Cervical, Cancer screening test, Cancer precursors, cervical intraepithelial neoplasia, Colposcopy, HPV test, HPV infections

Source Citation

Perkins RB, Guido RS, Castle PE, Chelmow D, Einstein MH, Garcia F, Huh WK, Kim JJ, Moscicki AB, Nayar R, Saraiya M, Sawaya GF, Wentzensen N, Schiffman M; 2019 ASCCP Risk-Based Management Consensus Guidelines Committee. 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors. J Low Genit Tract Dis. 2020 Apr;24(2):102-131. doi: 10.1097/LGT.0000000000000525. Erratum in: J Low Genit Tract Dis. 2020 Oct;24(4):427. PMID: 32243307; PMCID: PMC7147428.