Abnormal Papanicolaou Smear

Issues of Concern

A Pap test serves as a screening tool for detecting cellular changes in the cervix. Evidence documents variable sensitivity and specificity across different settings.[5] Because the test does not achieve 100% sensitivity, routine cervical cancer screening remains essential.[6][7] Furthermore, cervical cancer screening has also progressed beyond isolated Pap testing to incorporate HPV testing.[8]

From a societal perspective, racial disparities affect the timeliness of cervical cancer diagnosis. Localized cervical cancer diagnosis occurs in 47% of White individuals compared with 36% of Black individuals. Regional diagnosis occurs in 35% of White individuals and 41% of Black individuals.[3] Reduced rates of early detection among Black women contribute to diagnosis at more advanced stages of disease.

Causes

Human Papillomavirus Risks

The primary cause of abnormal Pap tests is associated with infection by HPV. HPV serotypes 16/18 are the most common strains that cause cervical cancer.[9] Both of these serotypes appear in 70% of cervical cancer cases. Other strains that increase risk for cervical cancer include HPV 45, 58, 51, 39, 66, and 73.[10] Lower-risk HPV serotypes include serotypes 6 and 11, but are more commonly associated with genital warts.[11] Please see StatPearls' companion resources, "Human Papillomavirus" and "Cervical Dysplasia," for further information.  

Smoking Risk Factors

Smoking has also been shown to increase the risk of cervical cancer.[12] The carcinogens in the smoke disseminate throughout the body and can be detected in cervical mucus. These carcinogens can alter the balance of oncogenes within these cells, resulting in cancerous growth. Research also shows that women who have a high number of full-term pregnancies are at an increased risk of developing cervical cancer.[13] 

Pharmacologic-Associated Cervical Cancers

Women who take oral contraception for prolonged periods have a temporary increase in the risk of acquiring cervical cancer.[14][15] Additionally, patients who have human immunodeficiency virus or are immunocompromised have an increased risk of acquiring HPV, which can lead to cervical cancer. Both of these reduce the ability of the immune system to clear the virus. From a more historical reference, children who had their mother take diethylstilbestrol (DES) during the pregnancy are at elevated risk of clear cell adenocarcinoma of the vagina and cervix.[16] DES was prescribed to pregnant women from the late 1930s until 1971 in an attempt to reduce the risk of miscarriage and premature labor; however, DES is no longer used in pregnancy due to a lack of proven benefit and the later recognition of significant adverse effects. Please see StatPearls' companion resource, "Clear Cell Carcinoma of the Cervix," for further information.  

Human Papillomavirus Vaccine Preventive Strategies

The HPV vaccination is a safe, highly effective, and long-lasting strategy for the prevention of cervical cancer and other HPV-related malignancies. HPV vaccination protects against multiple HPV types, including the high-risk oncogenic strains 16 and 18 that are responsible for the majority of cervical cancer cases.

HPV vaccination is most effective when administered before exposure to the virus; therefore, this prevention strategy is routinely recommended at 11 to 12 years of age, with initiation possible as early as 9 years of age. Individuals aged 9 to 14 years require a 2-dose schedule given 6 to 12 months apart, whereas those starting at 15 years or older, as well as immunocompromised individuals, require a 3-dose series over 6 months. Although routine vaccination is not universally recommended beyond 26 years of age, selected adults between 27 and 45 years may still benefit based on individual risk assessment. Please see StatPearls' companion resource, "Human Papillomavirus Vaccine," for further information.  

Extensive real-world evidence shows that HPV vaccination can prevent more than 90% of HPV-related cancers, with marked reductions in HPV infections, genital warts, and cervical precancerous lesions. Long-term follow-up studies demonstrate sustained protection for over a decade without evidence of waning immunity, highlighting its central role in the primary prevention of cervical cancer.

Anatomical Pathology

Cervical cancer screening requires acquiring cervical cells in the transitional zone of the cervix. The transitional zone is also called the squamocolumnar junction because this area is the junction between the ectocervix and the endocervix. The ectocervix is comprised of nonkeratinized stratified squamous epithelium, while the endocervix is simple columnar epithelium. The simple columnar cells also have glandular cells that secrete mucus.

Anatomic pathology in cervical cytology mainly arises from the 2 cell types found in the transitional zone: epithelial and glandular. Epithelial cell pathology is graded in the following ascending fashion:

• Atypical squamous cells of undetermined significance (ASCUS): This is the most common abnormal pap smear diagnosis.[17] An ASCUS diagnosis usually correlates with an HPV infection and can reflect precancerous changes in the cells, inflammation, or a response to trauma (see Image. Squamous Cells, Acute Inflammation). Please see StatPearls' companion resource, "Atypical Squamous Cells of Undetermined Significance," for further information.
• Atypical squamous cells cannot exclude high-grade intraepithelial lesions (ASC-H): This diagnosis is exceedingly rare, and colposcopy is required to confirm the absence of high-grade intraepithelial lesions. Please see StatPearls' companion resource, "Colposcopy," for further information. 

If the pathologist can identify a morphological change within the cells, the following nomenclature is used:

• Low-grade squamous intraepithelial lesions (LSIL): This term encompasses a spectrum of changes in squamous cells that may progress to squamous cell carcinoma. A finding of LSIL is highly suggestive of an HPV infection. This nomenclature includes cervical intraepithelial neoplasia (CIN) 1 and mild preinvasive cervical disease. While this finding occurs in 2% of all Pap smears, the 2-year regression rate of LSIL is over 85%.[18] Less than a quarter of LSIL findings will progress to higher-grade lesions.
• High-grade squamous intraepithelial lesions (HSIL): This term is a general term for abnormalities in squamous cells with a high risk of progressing to cervical cancer. This finding is highly suggestive of an HPV infection. This title includes the historical terminology of CIN2, CIN3, moderate preinvasive cervical disease, severe preinvasive cervical disease, and carcinoma in situ.[19] The ASCCP's preferred terminology for these classifications is cervical preinvasive disease. 
• Squamous cell carcinoma: This is rarely diagnosed on a pap test.[20] This condition only occurs with a significant progression of cervical cancer.

In addition to the epithelial cells, the pathologist can identify abnormal glandular cells, which are graded in the following ascending fashion:

• Atypical glandular cells (AGC): This cell type can be found in the endocervix and may also appear in other parts of the reproductive tract, eg, the endometrium. AGC is uncommon, diagnosed in 0.3% of all Pap smears. AGC identifies cells that have progressed beyond temporary inflammatory changes but have not yet mutated enough to be diagnosed as adenocarcinoma.[21] Although not a diagnosis of adenocarcinoma, a 61-fold increase in the risk of adenocarcinoma is observed compared with patients with a normal Pap test result.[22] A finding of AGC can result from benign etiologies, eg, cervicitis, oral contraception use, pregnancy, or endometrial polyps.[23] AGC is divided into 2 main categories: "undetermined significance" and "undetermined significance with a squamous abnormality." Please see StatPearls' companion resource, "Atypical Glandular Cells (AGS)," for further information. Squamous abnormalities can be found in 31% of AGC diagnoses and are more likely to appear in patients younger than the age of 35.[24] A diagnosis of AGC increases the risk of cancers throughout a woman's reproductive tract. ACG is also divided into the following subtypes based on the suspected origin of the glandular cells:
  • Atypical glandular cells favor endocervical origin (AGC-EC): This diagnosis is least likely to have associated endometrial cancer.[25]
  • Atypical glandular cells favor endometrial origin (AGC-EM): This diagnosis is most likely to have associated endometrial cancer.[25]
  • Atypical glandular cells favoring neoplasia (AGC-FN): This diagnosis was added in 2019 and carries a high risk of underlying malignancy, up to 50%.[26]
  • Atypical glandular cells not otherwise specified (AGC-NOS): This diagnosis is in the middle, having an association with endometrial cancer.[25]

• Endocervical adenocarcinoma in situ: This diagnosis has become more frequent in recent decades, with a prevalence of 11.2 per 100,000, and is most likely to be diagnosed in patients between the ages of 30 and 40.[27] Endocervical adenocarcinoma in situ (AIS) usually correlates with a squamous lesion. Like the other abnormal Pap smear findings, high-risk serotypes of HPV are a risk factor.

• Adenocarcinoma: This diagnosis is the invasive version of AIS, which can be found in the transition zone but is more commonly present within the cervical canal. This condition also derives from the endocervical glandular epithelium.[28] An increase in this diagnosis has been noted, even though cervical squamous carcinoma cases have decreased.
  • Adenocarcinoma malignum: A rare cervical disease that is diagnosable with a pap test. This disease usually arises in the endocervical region and is often misdiagnosed as a benign lesion.[29] Cytologic features include significant cytoplasm with ovoid nuclei.[30] Histologically, the cells are in a honeycomb pattern. The mucinous glands secrete copious fluid, leading to symptomatic vaginal discharge.

Less commonly, abnormal Pap smears can arise from other histological subtypes. Cervical cancer can arise from a neuroendocrine origin, with the 2 main subtypes being large and small cell. Other types of tumors that can cause cervical cancer include sarcoma, lymphoma, and melanoma.

Clinical Pathology

Abnormal Pap test results often present without symptoms, making targeted history-taking essential. Clinicians should inquire about bleeding between periods, changes in the menstrual cycle, abnormal vaginal discharge, and dyspareunia. Any increase in unexplained bleeding or menstrual bleeding warrants evaluation for possible cervical cancer. Postmenopausal bleeding raises particular concern for neoplasms, although such presentations are more commonly associated with endometrial cancer. In more advanced stages of cervical cancer, patients may report pelvic pain and dysuria.

Biochemical and Genetic Pathology

The most common etiology of abnormal pap smears is due to HPV infections. HPV is a nonenveloped, circular, double-stranded deoxyribonucleic acid virus. The high-risk HPV serotypes are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 because they make the oncoproteins E6 and E7.[31][32] Both of these oncoproteins result in the immortalization of the infected cervical cells. E6 binds to the intracellular transformation-related protein (p)53, causing it to degrade. The p53 protein is primarily utilized in the apoptotic pathway to upregulate the transcription of p53 upregulated modulator of apoptosis (PUMA) and phorbol-12-myristate-13-acetate-induced protein 1, NOXA, leading to cell death. E7 focuses on activating the cell cycle, allowing for replication.

In noninfected cells, one way the cell cycle becomes arrested in G1 is through the binding of Rb to E2F. E2F is a transcriptional activator needed to progress the cell into the S phase, but cannot do so when bound to Rb. In high-risk HPV strains, E7 binds to Rb, releasing E2F.[33] With E2F unbound from Rb, the infected cells are signaled to begin the S phase. Genetically, breast cancer gene (BRCA) mutations play a role in the susceptibility to developing cervical cancer. Its translational product is a tumor suppressor protein. BRCA1 and BRCA2 are integral in many pathways, but are especially important for deoxyribonucleic acid (DNA) repair. When double-stranded DNA breaks occur, these proteins help with homologous recombination repair. Without these proteins, genomic instability can lead to neoplastic changes.[34] Within the BRCA homologous repair pathway, other identifiable genes include BRIP1, RAD51C, RAD51D, PALB2, ATM, CHEK1, and CHEK2.

Morphology

The morphological changes recognized in abnormal Pap smears include multinucleation, hyperchromasia, and perinuclear cytoplasmic vacuolization. The nuclei of the cells can also become enlarged, up to 3 times the size of a normal cell nucleus. Other nuclear features include increased keratinization but an absence of nucleoli. These changes that squamous cells undergo are categorized as koilocytosis.

Mechanisms

A Pap smear involves the collection of cervical cells using a brush inserted into the cervical os. The brush should extend beyond the visualized portion to improve sampling of the transition zone. Alternative collection devices include a spatula or broom. Two methods exist for analyzing collected cells: liquid-based cytology, eg, ThinPrep and SurePath, and conventional cytology. Cytology includes 2 subtypes.[35] One subtype allows the collection device to be detached and retained with the sample for analysis, while the other requires agitation of the collection tool within the medium because the device does not accompany the sample.

Liquid-based cytology preserves cells in a buffered alcoholic solution containing 25% to 55% methanol, which acts as a fixative and supports storage for 3 to 4 weeks. ThinPrep, manufactured by Hologic, represents the most commonly used system in the United States.[36] Instructional guidance recommends minimizing the use of gel-based lubricants before specimen collection, as these substances can adhere to the filter membrane and produce inaccurate results.

Evidence suggests that liquid-based cytology may improve the detection of glandular pathology. Conventional cytology involves direct transfer of collected cells from the brush onto a slide, which defines the Pap smear technique. During transfer, minimizing drying artifacts from prolonged air exposure remains important. According to ACOG, both liquid-based and conventional cytology demonstrate equivalent effectiveness in cervical cancer screening.

Clinicopathologic Correlations

AGC in patients with HPV serotypes 16 and 18 has an increased risk for AIS and warrants closer surveillance. When AIS occurs concurrently with LSIL, clinical management should proceed according to an AIS diagnosis.

Clinical Significance

The Papanicolaou test is an effective screening test that detects cervical cancer as well as cellular changes that have the potential to develop into cervical cancer. Any diagnosis of an atypical pap smear results in more frequent and further testing. Management guidelines for abnormal Pap test findings vary across different professional societies and groups. The following management strategies, according to ACOG, are currently recommended for each abnormal result:

• ASCUS: The management of ASCUS depends on the age of the patient. For patients aged 21 to 24, repeat the Pap smear in 1 year if HPV status is positive or unknown. Performing a reflex HPV test on the same pap smear sample is also acceptable. HPV status will determine future Pap smear frequencies.
  • If HPV is negative, then the patient is recommended for follow-up in 3 years.
  • If the follow-up Pap smear continues to show ASCUS, then an additional Pap smear in 1 year is recommended.
  • For patients between 25 and 29, screening is still the same if they have a negative HPV test. However, if they have positive HPV, colposcopy is recommended instead of waiting 1 year for a follow-up pap smear. If ASCUS persists on the follow-up Pap smear, the next step is colposcopy.
  • For patients between 30 and 65 years of age, a more significant concern is the progression of cervical cancer. If ASCUS is documented on a pap smear and HPV is negative, following the same management as the other age groups is recommended. If HPV status is positive or unknown, a repeat pap smear is recommended within 4 months. 
• LSIL: Repeat Pap smear in 1 year.
• ASC-H: Immediate further testing with colposcopy is recommended.
• HSIL: Further testing with colposcopy is recommended.
• AGC: Further testing can include endocervical sampling and an endometrial biopsy to source the location of the cells. Obtaining a targeted family history at this time is essential. The patient should have their HPV status evaluated.
• AIS: Immediate diagnostic excision procedure along with an endocervical and endometrial biopsy. The diagnostic excision will help confirm the diagnosis and determine the extent of the invasion.
• Squamous cell carcinoma: Immediate excision and further workup are necessary.