Introduction

"Atypical glandular cells (AGC)" is the term adopted by The Bethesda system of reporting cervicovaginal cytology.[1] The Bethesda reporting system was initiated for uniform reporting and overcame interobserver variability of cervicovaginal cytology in 1988.[2][1] 

One of the major revisions of the Bethesda system is the substitution of “Atypical glandular cells of unknown significance (AGUS)” with “Atypical glandular cells (AGC).”  AGC is the term used to categorize glandular cells with cytologic features that are too pronounced to be called inflammatory or reactive but cannot be classified as malignant (endocervical adenocarcinoma in situ or invasive). AGC is a significant diagnosis and can include conditions that range from reactive and inflammatory conditions to dysplasia and malignancy.[2][3] This carries a great deal of clinical significance, especially on younger patients, where the management is predominantly based on the cytologic diagnosis.

Issues of Concern

Endocervical adenocarcinoma is the second most frequent carcinoma of the cervix after Squamous cell carcinoma (SCC).[4] Endocervical adenocarcinoma accounts for about 15 to 20% of cervical cancers. The incidence is reported to be on the rise, and this is thought to be mainly because of the decreasing incidence of invasive SCC due to early recognition of these lesions by cytology screening. Carcinoma of the cervix accounts for approximately 10% of cancer diagnosis worldwide and approximately 8.5% of cancer-related deaths. Though the incidence of endocervical adenocarcinoma is less than squamous cell carcinoma of the cervix, it is associated with a worse prognosis. It is more predominant in developing countries. In the US, the adenocarcinoma of the cervix has increased in incidence and is associated with increased numbers of sexual partners, younger age of initiation of sexual activity, and nulliparity.[5] 

Endometrial carcinoma is a malignancy that is more common in the developed part of the world. It mainly affects postmenopausal women. More than 65,000 cases are diagnosed in the US yearly. It is the sixth most common cancer in women worldwide.[6] The mean age of presentation is 60 years. Usually present at an early stage as post-menopausal metrorrhagia. 

Atypical glandular cells can also be reported in numerous reactive, inflammatory, and metaplastic conditions in addition to the above two malignancies. In these conditions, the cytologic features are too pronounced to place them under a benign category.

Causes

The AGC category includes dysplasia of the endocervical as well as endometrial cells. The causes, however, for these two conditions are different.

About 80% to 95% of endocervical adenocarcinomas are associated with HPV subtypes. Usual-type adenocarcinoma of the cervix is related to high-risk HPV subtypes like HPV-16 or HPV-18. HPV accounts for 50-58% of endocervical adenocarcinomas. Endocervical adenocarcinoma- mucinous,  gastric type is commonly seen in Japan and is associated with HIK1083 and MUC6 immune profile expression and germline inactivation of LKB1 seen in patients with Peutz-Jegher’s syndrome (also called adenoma malignant). Mucinous carcinoma, intestinal-type, is a rare type that shows 85% positivity to MUC-2 and is found to be associated with HPV.[4] In HPV-associated cancers, the risk of malignancy is dependent on the viral burden.[7]

Endometrial carcinomas are associated with unopposed exposure to estrogen. There is an increased incidence with increased duration and dose of exposure. Obesity is an important contributing factor.[8] Other factors, like nulliparity and anovulation, are also implicated in the development of these cancers.  

A huge number of other factors are associated with AGC apart from the other two conditions. For example, an intrauterine device (IUD) can cause significant changes in the endocervical cells. In the absence of a history of IUD or if the IUD status of the patient is unknown, these patients are likely placed in the AGC category. Severe inflammation, vigorous sampling, and radiation can induce changes that might mimic dysplasia and are often placed in the AGC category. Getting adequate, relevant clinical history is of extreme importance in these patients.

Anatomical Pathology

The Bethesda system uses criteria like nuclear crowding, nuclear overlap with or without pseudo-stratification, enlarged nuclei (2-3 times the size of normal endocervical cells), variation in nuclear size and shape, hyperchromasia, chromatin irregularity, presence of nucleoli, mitoses, increased nuclear-cytoplasmic ratio, and distinct and discernible cell borders. Atypical glandular cells – according to the Bethesda system of 2015- “should be categorized according to the site of origin as endocervical or endometrial whenever possible”. If the site of origin cannot be categorized then the general term "atypical glandular cells" can be used.

The glandular cells are classified according to the site of origin into the following subcategories (Bethesda 2015).[2][1]        

•  Atypical:
  • Endocervical cells (NOS or specify in comments)
  • Endometrial cells (NOS or specify in comments)
  • Glandular cells (NOS or specify in comments)

• Atypical
  • Endocervical cells, favor neoplastic
  • Glandular cells, favor neoplastic

Atypical Glandular Cells, NOS:

Definition: Cells resembling endocervical cells with nuclear atypia, that exceeds a reactive or reparative process but lack unequivocal features of endocervical adenocarcinoma in situ or invasive adenocarcinoma (Bethesda 2015). Whenever possible the location of origin of cells has to be specified eg: " endocervical" or " endometrial".

Criteria:

• Cells occurring in sheets and strips, with crowding, overlapping nuclei, and pseudo-stratification
• Enlarged nuclei, up to about 3- 5 times the normal endocervical cells
• Minor variation in nuclear size and shape
• Mild nuclear hyperchromasia
• Mild irregularity of chromatin
• Rare nucleoli
• Rare mitotic figures
• Abundant cytoplasm with a fairly increased nuclear-cytoplasmic ratio
• Distinct cell borders

Criteria for reporting Atypical Endocervical cells, Favor neoplastic:

Definition: Glandular cells with morphology, either quantitatively or qualitatively falls short of interpreting as endocervical adenocarcinoma in situ or invasive adenocarcinoma (Bethesda 2015).

Criteria:

• Cells occurring in sheets and strips, with nuclear crowding, overlap, and/or pseudo-stratification
• Cell groups resembling rosettes/ or forming glands or feathering
• Enlarged and elongated nuclei with hyperchromasia
• Coarse chromatin with heterogeneity
• Occasional mitoses and/ or apoptotic bodies
• Increased nuclear-cytoplasmic ratio
• Ill-defined cell borders

Criteria for reporting Atypical Endometrial cells:

DEFINITION: The distinction of atypical endometrial cells from benign endometrial cells is mainly based on the increased nuclear size in the atypical cells. The atypical endometrial cells are not generally further classified as " favor neoplastic" as it is a tough call and often gives poorly reproducible results. ( Bethesda 2015)

Criteria:

• Cells in small groups, usually 5 to 10 per group
• Slightly enlarged nuclei in comparison to normal endometrial cells
• Mild hyperchromasia
• Chromatin heterogeneity
• Occasional prominent nucleoli
• Scant vacuolated cytoplasm
• Ill-defined cell borders

Clinical Pathology

The American Cancer Society has issued the following guidelines (2020) for cervical cancer screening:[9]

• Cervical cancer screening should begin at age 25 years.
• Women aged 25 to65 years should have a primary HPV test every 5 years. If primary HPV testing is not available, screening should be done with either co-testing (pap smear and HPV DNA) every 5 years or pap test alone every 3 years.
• Women aged >65 years who have had regular screening appointments in the past 10 years with no history of serious diagnosis in the past 25 years can stop screening.
• Women who underwent hysterectomy with removal of uterus and cervix, except if the hysterectomy was done for cervical cancer can stop screening. Women whose cervix was not removed should undergo regular recommended cervical cancer screening.

A quadrivalent HPV vaccine has been approved in the United States and Europe, is effective against 4 HPV subtypes (6, 11, 16, and 18), and is reported to be effective in preventing cervical cancer.[10] Recently a second-generation vaccine was approved for use and has shown to be effective against approximately 90% of the HPV subtypes causing cervical cancers.[11] Second generation HPV vaccine along with recommended screening with cervical cytology and high-risk HPV DNA testing offers the maximum available protection against cervical cancer.

The USPSTF (United States preventive services task force)'s recommendations for cervical cancer screening, published in 2018, include[12]:

1. Screening with cervical cytology alone every 3 years in women aged 21 to 29 years
2. Every 3 years with cervical cytology alone or every 5 years with cervical cytology and high-risk HPV combined testing or every 5 years with high-risk HPV testing in women 30 to 65 years old.[12][13]

The USPSTF recommends against screening for cervical cancer in women younger than 21 years and >65 years who have had prior normal cervical screenings and are not otherwise at high risk for cervical cancer.[13] The USPSTF recommends against screening for cervical cancer in women who underwent a hysterectomy with removal of the cervix and no prior history of cervical cancer or high-grade lesion (CIN 2 or 3).

Biochemical and Genetic Pathology

HPV infection is a significant risk factor for developing endocervical adenocarcinomas.[7] HPV is a DNA virus capable of penetrating through the basal cell layer of the cervical mucosa. HPV surface proteins bind to integrins, a membrane receptor in the epithelial cells. A secondary receptor might be involved in stabilizing the HPV in the cell and integration with host DNA. The HPV DNA replicates in the host cells and reaches the surface as the basal cells mature. Host cell factors are required for HPV replication and proliferation. The interaction of host cell factors with the LCR region of the HPV DNA leads to transcription of E6 and E7 proteins: E6 and E7 inactivate tumor suppressor genes and cyclin-dependent kinases. HPV E6 binds to P53 and leads to its increased ubiquitination while E7 binds to the Rb tumor suppressor gene, the final outcome of these interactions in increased DNA synthesis and cell proliferation. More damaged DNA increases over time, leading to cumulative accumulation of mutations and finally cervical dysplasia and malignancy.[3][14][15]

Endometrial carcinoma is classically divided into two subtypes (type 1 and type 2) related to clinical characteristics. Type 1 includes endometrioid and mucinous adenocarcinoma and is associated with increased exposure to estrogen and PTEN, KRAS, PAX2 gene alterations; type 2 includes poorly differentiated subtypes (clear cell, serous, undifferentiated, and carcinosarcomas) with aggressive clinical outcomes and often associated with TP53 alteration.[6]

Molecular classification of endometrial carcinoma according to The Cancer Genome Atlas (TCGA), divides it into four prognostic groups from best to worst prognosis:

1. POL-E: a group with polymerase ε exonuclease domain mutations
2. MSI: endometrial carcinoma with microsatellite instability
3. Copy number low (CNL)
4. Copy number high (CNH)[16]

Molecular analysis by microarray revealed the upregulation of CEACAM5, TACSTD1, S100P, and MSLN, with the expression of CEACAM5 and TACSTD1 associated with worse survival outcomes in endocervical and uterine adenocarcinoma.[17]

Morphology

Endocervical adenocarcinoma:

1. Usual type adenocarcinoma: HPV related. There is the proliferation of glandular or papillary structures, loss of polarity, overlapping of cells, and increased mitotic activity.
2. Mucinous adenocarcinoma – gastric type: This type is predominantly composed of a proliferation of pyloric glands. Usually associated with poor prognosis.
3. Mucinous adenocarcinoma – intestinal type: associated with the proliferation of glands with goblet cells or sometimes Paneth cells.
4. Mucinous adenocarcinoma- signet ring cell type: Predominant signet ring cell morphology.
5. Villoglandular adenocarcinoma- carries an excellent prognosis. Frond like papillary growth pattern. Associated with HPV subtypes 16, 18, and 45.[4]
6. Endometrioid adenocarcinoma – resembles endometrial adenocarcinoma. It is also thought that this type might develop from endometriosis.
7. Clear cell adenocarcinoma – consists of cells with clear or eosinophilic cytoplasm, which sometimes show hobnailing with moderate pleomorphism and rare mitoses.[18] 
8. Mesonephric adenocarcinoma- rare type, derived from remnants of mesonephric ducts. Tubular structures are predominant, with hyaline sections, there is mild-moderate pleomorphism among the cuboidal cells lining the tubules.[19]
9. Serous adenocarcinoma- consists of broad irregular papillae lined by cuboidal cells. There is a higher degree of nuclear pleomorphism and intense staining of tumor cells with WT-1 and P53 immunohistochemical stain.[20]

Endometrial adenocarcinoma:

There are two types, type 1 and type 2. The histotypes are endometrial, mucinous, clear cell, serous, undifferentiated, and carcinosarcomas.

Clinicopathologic Correlations

Endocervical adenocarcinoma is usually seen in relatively young females. The symptoms are usually non-specific, like spotting, pain during intercourse, and persistent pelvic pain. This is usually seen in women with a history of multiple sexual partners, especially HPV associated types. Most of the cases are diagnosed on routine cervical screening.

Abnormal uterine bleeding (AUB) is the most common symptom seen in post-menopausal women, especially in endometrial carcinoma. Weight loss, anemia, vaginal discharge are the other non-specific symptoms of endometrial carcinoma. Radiologic evaluation in patients with AUB might reveal the increased thickness of endometrium in patients with endometrial carcinoma on Ultrasound examination. Usually, the endometrial thickness of >10mm is thought to be worrisome for endometrial carcinoma; however, endometrial carcinoma cannot be ruled out for endometrial thickness <10mm. An endometrial stripe, polyp, or any other irregularity on ultrasound should raise concern for endometrial carcinoma.

Management

Atypical glandular cells (AGC) diagnosis should be immediately followed up with a clinician. There is risk of premalignant lesions in patients diagnosed with AGC is as high as 11%, the risk of endometrial cancer is 3%, and the risk of cervical cancer is 1%. AGC is found in <1% of cervical cytology specimens. About 50% of the patients diagnosed with AGC will be found negative on colposcopy and endometrial sampling. Management is mainly based on the type of AGC diagnosis.  Squamous lesions are most commonly found in patients with AGC diagnosis. When there is a malignancy of glandular cells, it is most commonly from the endocervix or endometrium. However, in rare cases, malignancy from the ovary, breast, fallopian tubes, pancreas, and colon have also been reported. The management differs according to the diagnosis and also pregnancy status.

The ASCCP (American Society for Colposcopy and Cervical Pathology) issued the 2019 consensus guidelines. 

Initial management of all categories except atypical endometrial cells is colposcopy with endocervical sampling. Women 35 years or older who are at risk for endometrial carcinoma should also undergo endometrial sampling. Women <35 years should undergo endometrial sampling if they are at higher risk for endometrial carcinoma (abnormal uterine bleeding, obesity, or conditions suggesting chronic anovulation).

• AGC, NOS: after a negative colposcopy and endocervical sampling, these patients should be followed-up with cytology at 12 and 24 months along with high-risk HPV testing. Suppose negative should be co-tested every 3 years. If any abnormality is detected, colposcopy should be done.
• AGC favor neoplasia: initial colposcopy and endometrial sampling, if negative, should be followed by cone biopsy.  A negative colposcopy and endometrial biopsy do not exclude the diagnosis in these patients.[1]
• In patients with Atypical endocervical cells, "intact specimen with interpretable margins should be provided, concurrent endocervical sampling above the excision bed is preferred.
• Those with atypical endometrial cells should undergo endometrial as well as endocervical sampling. Colposcopy may be delayed if endometrial pathology is identified in these patients. 
•  For pregnant patients with AGC diagnosis endocervical canal can be gently brushed with a cytobrush. Biopsies and curetting should be avoided as there is a risk of causing harm to the pregnancy. Non-pregnant patients with AGC, endometrial, and endocervical sampling should be done, with or without colposcopy. If the sampling is negative for any pathology, then a colposcopy should be considered. 
•  Failure to find a lesion (based on strong clinical suspicion) should be followed by further studies like transvaginal ultrasound, CA-125 levels, and CA19-9, as sometimes the diagnosis of AGC is associated with an occult primary in other organs.

Subsequent management depends on the results of initial sampling and cytologic interpretation. Repeat cytology is not an option, as AGC is a high-risk category, and these patients may harbor indolent preinvasive or invasive carcinomas. Excisional sampling is recommended if the initial management fails to provide a diagnosis. In women diagnosed with Atypical glandular cells, NOS, if the initial workup reveals no evidence of carcinoma >CIN2+, Adenocarcinoma in situ, or carcinoma, co-testing at 12 and 24 months is recommended. If the co-testing at 12 and 24 months is negative, these patients can be managed with repeat co-testing every 3 years. If any abnormality is noted, colposcopy is recommended.

Clinical Significance

HPV testing provides a reliable prediction of disease incidence and progression and allows considerably longer screening intervals after a negative HPV molecular test than after a negative cytology test. In the ARTISTIC cohort of 24,496 women with 10-year follow-up, about three-quarters of the HPV+ with normal cytology cleared their infections within about 3 years. Their risk of developing a CIN3 lesion within this time frame is considered very low (1.5%). The cumulative risk of developing CIN3 in women with type-specific persistent infections is six times higher than in new infection cases.[21]

The diagnosis of AGC includes the following differential diagnosis:

1. Inflammation
2. Metaplasia
3. Radiation changes
4. Arias-Stella reaction
5. Microglandular hyperplasia
6. Oral contraceptives
7. Intrauterine device
8. Endocervical adenocarcinoma
9. Endometrial adenocarcinoma
10. Squamous intraepithelial lesion

Prognosis

Though the management of patients with SCC and endocervical adenocarcinoma is similar, the Kaplan- Meier survival analysis showed worse outcomes in patients with adenocarcinoma.[22][4]  The HPV associated endocervical adenocarcinoma carries a better prognosis than mucinous type – gastric endocervical adenocarcinoma, with a 5-year survival rate of 77% when compared to mucinous type gastric, which is 30%.[4] The villoglandular form of endocervical adenocarcinoma carries an excellent prognosis. For endocervical adenocarcinoma, younger patients (<35 years) had a better prognosis than patients >65 years. Higher the stage at the time of diagnosis, lymph node metastases, lymphovascular invasion, higher depth of stromal invasion, and higher grade were associated with poor prognosis.[23]

The prognosis of endometrial adenocarcinoma is mainly based on the stage of the disease. The international federation of obstetrics and gynecology (FIGO) has developed a grading system. Grade 1 tumors exhibit predominantly glandular morphology with <5% solid growth. Squamous differentiation is absent. Grade 2 tumors have solid growth between 6- 50%, and Grade 3 shows solid growth >50%. Higher myometrial invasion is also associated with a worse prognosis.[24] 

Complications

The diagnosis of Atypical glandular cells includes a wide range of clinical conditions from reactive conditions due use of IUD, prior radiation therapy, etc. to malignant neoplasms of the endocervix and the endometrium. However, rarely AGC might also represent malignant neoplasm of the ovary and the fallopian tubes. In cases that are mainly reactive, it can be considered an overcall to diagnose them as AGC, whereas the diagnosis of atypical cells arising from the ovary and the fallopian tube would lead to subsequent colposcopy and endometrial sampling and might be at risk for missing the diagnosis.

Post-operative and Rehabilitation Care

Patients managed with colposcopy with endometrial sampling might experience pain or discomfort. The colposcopy is an office procedure typically lasting between 10-20 minutes. There might be some bleeding. Vaginal pads may be used to catch any bleeding. Tampons, douching, and vaginal intercourse should be avoided for at least a week or as clinically indicated. This procedure is associated with low morbidity.

There might be anxiety associated with the diagnosis, procedure, and waiting for results. Appropriate counseling of patients might help reduce anxiety, and defining a clear timeline would help alleviate some of the anxiety. The risks and benefits of the procedure should be clearly explained to the patient, and complications and the rate of complications should also be explained. Brochures or pamphlets should be provided to the patient, and also appropriate websites where they can find information should be given.

Summary

• Cervical cancer screening is a powerful tool and has greatly impacted the outcomes of cervical cancer in women.
• Atypical glandular cells include a wide variety of conditions, which range from reactive conditions to dysplasia to carcinoma.
• AGC is the term used when the nuclear features are more worrisome than usually seen in reactive conditions, but not enough to call it malignant
• More than 50% of the women diagnosed with AGC have no pathology on colposcopy and subsequent endometrial sampling.
• In patients with “AGC, favor neoplastic,” if the colposcopy and endometrial sampling are negative, it does not exclude malignancy. A cone biopsy is the next recommended step, and it is the responsibility of the treating physician to conduct a thorough examination and diagnostic work-up

Enhancing Health Care Team Outcomes

Clear communication and effective management are of extreme importance. The diagnosis of AGC requires diligent examination and follow-up. The patient should be clearly explained how strict follow-up might result in better and favorable outcomes. Advocating for HPV vaccination might further decrease the diagnosis of these cancers.