Benign Proliferative Reactions, Intraepithelial Neoplasia, and Invasive Cancer of the Uterine Cervix

Key Features Parabasal Cells ( Fig. 8-3 ).

• More common in low-estrogen and/or progesterone-dominant states (postpartum, postmenopause, progestin-based contraception)

• Round to oval

• Commonly in sheets

• Increased nuclear to cytoplasmic ratio

• Moderately dense, distinct, cyanophilic cytoplasm

• Nuclei with evenly distributed chromatin and conspicuous nucleolus.

Epithelial regeneration in the squamous epithelium is thought to take place in the basal layer, composed of a single layer of primitive appearing cells with scant cytoplasm, oval to round nuclei, and relatively prominent nucleoli. Under normal conditions, true basal cells are not seen in cytology smears unless there has been some type of injury allowing for the complete removal of the epithelium. Parabasal cells, accordingly, are also unusual in smears from reproductive aged women unless certain pathologic or medically induced processes are present. The reduction in estrogen during the peri- and postmenopausal years make parabasal cells common in smears from this age group. Low-estrogen states such as in progestin-only birth control, childhood, the post-partum period, and certain amenorrheic states can cause a similar cytologic picture. In cytology specimens, parabasal cells are round to oval and have a small amount of moderately dense, distinct, cyanophilic cytoplasm, oval to round nuclei, and, similar to basal cells, can have relatively prominent nucleoli.

Key Features of Intermediate Cells ( Fig. 8-4 ).

• Most common cell type during reproductive age

• Abundant lightly cyanophilic cytoplasm, oval

• Nucleus round to oval, used as comparative quality and size marker: 8 to 10 µm, sometimes larger (e.g., perimenopause, progestin contraception)

• Evenly distributed, open, finely granular chromatin

• Smooth nuclear membrane.

A major part of the thickness of the epithelium during reproductive years is formed by the intermediate cell layer. With maturation toward the surface from the parabasal layer, the intermediate cells become better differentiated, with an increase in cytoplasmic volume. The cytoplasm of a normal intermediate cell is lightly cyanophilic; the nucleus is round to oval and slightly reduced in size to about 8–10 µm, with a clearly defined, smooth nuclear membrane and evenly distributed, finely granular chromatin. The normal intermediate cell serves as a size and quality standard for comparison during evaluation of pathologic processes, especially those due to HPV.

Key Features of Superficial Cells ( Fig. 8-5 ).

• Most common cell type in high estrogen states

• Large, polygonal cells with abundant lightly eosinophilic, translucent cytoplasm

• Sharply defined cytoplasmic membrane

• Small nucleus (3–5 µm) with dense, evenly distributed chromatin

• Smooth nuclear membrane

The superficial squamous cells are large (approximately 40 µm) and polygonal, representing the end-stage in maturation of non-keratinized stratified squamous epithelium. These cells have less cohesive desmosome attachments, making them conducive to constant exfoliation from the mucosal surface. The cytoplasm is translucent, eosinophilic, and has sharply defined cytoplasmic membrane. The nucleus is small (3–5 µm), with dense, evenly distributed chromatin and a smooth nuclear membrane.

The entire maturation cycle of normal squamous epithelium is variable in length, depending upon a number of factors. As described in greater detail later, this process can be accelerated significantly under estrogen stimulation, such as during late pregnancy, the proliferative phase of the menstrual cycle, with exogenous estrogen therapy, or as a result of estrogenic states such as obesity and cirrhosis. As a result, there is exaggeration of the superficial layers of the squamous epithelium and hypersecretion in the endocervical columnar epithelium. Reduced maturation, conversely, is seen during low-estrogen and progesterone/progestin-predominant states such as luteal phase of the menstrual cycle, post-partum, menopause, childhood and adolescence, and during exogenous progestin therapy. In such states, intermediate cells line the epithelial surface and are exfoliated in cytology specimens as the characteristic navicular cell. In reduced maturation states, cytolysis may be conspicuous, resulting in a large number of bare nuclei in cytologic preparations (see below, Fig. 8-7 ). Although generally not followed in routine cervical screening today, the ideal time for collection of a screening cervical cytology specimen is considered to be between menstrual cycle days 7 and 14, as obscuring menstrual material is usually not present, superficial/mature squamous cell predominate, and dysplastic cells are most easily recognizable against this mature squamous background.

Key Features of Normal Endocervix ( Fig. 8-6 )

• Sheets and strips of columnar cells with organized “honey­comb” architecture

• Apical, transparent, weakly cyanophilic, variably vacuolated, fragile cytoplasm

• Bare nuclei can be seen

• Round to elongate nuclei

• Finely granular, evenly distributed chromatin with one or two chromocenters

• Variable nuclear size, multinucleation, ciliation, prominent nucleoli in reactive states

• Groups may be associated with reserve cells.

The endocervical mucosa is composed of a single layer of tall columnar cells that can have a pseudostratified appearance. The endocervical surface is not perfectly smooth, and contains irregular projections and invaginations extending to a variable depth into the cervical stroma depending on age and parity. The tangentially cut invaginations are often referred to as “endocervical glands,” even though glandular structures are not formed in the strict sense of the term. The endocervical cells produce mucus that covers the epithelium and varies in amount and consistency with hormonal status. On cytologic preparations, endocervical cells form small, flat, honeycomb-patterned sheets and strips. The apical orientation of the cytoplasm is maintained and is faintly cyanophilic, transparent, and can be variably vacuolated. Apical cilia may be present. Bare nuclei due to lysis of the fragile cytoplasm can be seen. The nuclei are round to elongate, vary in size, and have finely granular, evenly distributed chromatin. One or two small chromocenters are usually present. Variation in nuclear size, multinucleation, increased ciliation, and prominent nucleoli are common, even in minimally reactive states, and these features can become alarmingly prominent. So-called “reserve cells,” thought to represent the multipotent stem cell of the cervix, can be seen in histologic sections beneath the columnar cell layer, and are occasionally seen in cytologic preparations when the endocervix is under the influence of a proliferative stimulus.

Key Features of Atrophy ( Figs 8-7 , 8-8 )

• Parabasal cells predominate

• Cells in syncytial sheets or lying singly

• Streaming nuclei are oriented in a similar direction

• Increased nuclear to cytoplasmic ratios

• Nuclei with dark, smudgy, evenly distributed chromatin

• In atrophic vaginitis ( Fig. 8-9 ), inflammation and reactive squamous changes may be superimposed, in some cases simulating squamous cell carcinoma

  • Enlarged nuclei

  • Variation in nuclear size

  • Coarse chromatin with prominent nucleoli

  • Keratinization

  • Inflammation.

  

Atrophy of the cervical and vaginal squamous mucosa is a sometimes symptomatically irritating but inevitable physiologic process that occurs with menopause. Similar changes occur with hypoestrogenic states, such as during lactation and with high-dose progestin therapy. A classic atrophic smear in itself usually does not cause significant diagnostic problems, but atrophy can be a source of morphologic pitfalls indistinguishable from a high-grade squamous intraepithelial lesion (HSIL) and squamous carcinoma in some situations. The classic atrophic smear contains syncytial groups of parabasal-type cells with streaming nuclei oriented in a similar direction. The cells have high nuclear to cytoplasmic ratios and dark, smudgy chromatin. Nucleoli are usually absent. In cases of atrophy with inflammation (atrophic vaginitis), cell changes due to infection, epithelial injury, or degeneration can cause diagnostic problems. The nuclei can become enlarged, variable in size, and can contain chromatin that is coarsely granular and hyperchromatic. If the epithelium is ulcerated, regenerative/reparative cells can have very large nuclei with prominent nucleoli. Additionally, these reparative cells can become keratinized, creating a cytologic picture similar to squamous cell carcinoma (SCC). Prior to the advent of HPV testing, such smears were especially problematic to differentiate from high-grade neoplasia or cancer, and a short course of topical estrogen was recommended to induce maturation and improve the interpretability of the cytology. Epithelial dysplasia does not react in the same way to an estrogenic stimulus, and therefore would be expected to persist. Such smears can now be placed in an atypical cytology category when the diagnosis is unclear, with HPV testing adding useful additional information to guide management in most cases.

Key Features of Hyperkeratosis ( Fig. 8-10 )

• Anucleate, polygonal cells lying singly or forming overlapping, thick sheets

• Orangeophilic, yellow, or pink cytoplasm

• Can have keratohyalin granules

• Nuclear ghosts.

The squamous mucosa of the ectocervix is normally non-keratinized, but has the ability to become keratinized as a protective mechanism when under the influence of chronic irritative factors. Chronic irritations such as due to uterine prolapse or inflammation cause an increase in epithelial thickness (acanthosis), followed by the development of a granular layer and several layers of keratinized squamous cells, similar to the cutaneous surface. This formation of keratinized layers over an otherwise normally unkeratinized mucosal surface is termed “hyperkeratosis.” Clinically, hyperkeratosis may be visible as a white patch or “leukoplakia.” On cytologic preparations, hyperkeratosis is recognized by the presence of numerous anucleate, orangeophilic to yellow or pink polygonal cells in sheets or lying singly. Remnants of nuclei may be visible as a central clearing or “nuclear ghost.” Granular cell layer cells may also be present resembling intermediate or superficial squamous cells, but containing eosinophilic or cyanophilic keratohyalin cytoplasmic granules.

Key Features of Parakeratosis ( Fig. 8-11 )

• Sheets of orangeophilic to eosinophilic cells

• Dense cytoplasm

• Dark nuclei with variation in size and shape

• Can have keratohyalin granules

• Features overlap with low-grade HPV-associated lesions.

Parakeratosis is another protective reaction, and is characterized by a variable number of layers of keratinized nucleated squamous cells that are sharply demarcated from the underlying superficial zone. In cytology specimens, parakeratotic cells are either isolated or in sheets and resemble small superficial squamous cells. The cytoplasm, however, is more dense than a normal superficial squamous cell, and the nuclei can be more hyperchromatic with variation in size and shape. Para­keratosis can be particularly problematic in cytology specimens, because the morphology not only overlaps with low-grade squamous lesions ( Fig. 8-12 ), but can also physically mask an underlying dysplastic lesion that remains unsampled in the collection. For this reason it has generally been recommended that the person collecting the cytology specimen first scrape hyperkeratotic or parakeratic squamous mucosa to remove the keratin layer and then sample the underlying squamous mucosa, but this recommendation is based upon conflicting data regarding the false-negative rate for Papanicolaou tests containing hyperkeratosis/parakeratosis.

Key Features of Reserve Cells

• Small clusters or clinging to endocervical cells

• Scant (or absent), finely vacuolated, ill-defined cyanophilic cytoplasm

• Small, uniform, round to elongate nuclei

• Finely granular, evenly distributed, sometimes dark chromatin

• May have longitudinal nuclear grooves.

As previously alluded to, much controversy surrounds the origin, role, and even the very existence of reserve cells, especially as related to squamous metaplasia and HPV infection. Traditionally reserve cells are thought to be seen as one or two layers of primitive cells underlying the normal endocervical epithelium near the squamocolumnar junction. Hypotheses of origin have included various ideas ranging from ingrowth of germinative basal cells from the adjacent stratified squamous epithelium, to origin from fetal rests, to origin from endocervical stromal cells. Some have even suggested origin from the mature columnar cells in a process termed “prosoplasia,” but evidence to definitively point to any of these origins is lacking. For the purpose of interpreting cytology specimens, the origin of reserve cells is probably not as important as recognizing the morphologic features of these mysterious cells and distinguishing them from neoplastic processes.

Reserve cells are uncommon in cervical cytology specimens, and are usually arranged in the form of sheets or small clusters. Cytoplasm is cyanophilic, scant (or absent) and ill-defined with fine vacuolation. Cell borders are poorly defined, giving the aggregates the appearance of a syncytium. Reserve cells can be seen clinging to a group of columnar endocervical cells. Nuclei are small, relatively uniform in size and shape; the nuclei may be elongated and show longitudinal grooves. Chromatin is finely granular, comparable to the nucleus of an endocervical columnar cell. In reparative states, chromatin can become coarser, but nucleoli are generally not present. Polarity is generally maintained, however, helping to distinguish reserve cells from high-grade squamous dysplasia or adenocarcinoma in situ (AIS), for which there can be significant morphologic overlap, and even coexistence.

Key Features of Immature Squamous Metaplasia ( Fig. 8-13 )

• Isolated cells or occasionally sheets

• Variable cell size

• Dense, cyanophilic, often vacuolated cytoplasm

• Variably large nuclear size and increased nuclear to cytoplasmic ratio

• Slight irregularity in nuclear size, shape, and nuclear membrane.

Immature squamous metaplastic cells are more common in cervical smears, and are more often seen as isolated cells, correlating with the degree of maturation of the parent epithelium. Sheet-like aggregates do occur, and cell borders are usually distinct. Depending on orientation, the cells are round to oval, with the number of polygonal cells increasing with maturation. The cytoplasm of immature squamous metaplastic cells is dense, cyanophilic, homogeneous, and sometimes vacuolated. Vacuolation commonly occurs in the context of inflammation or as a consequence of degeneration, as in cytolytic smears in progesterone/progestin predominant states. Nuclei are variably large, creating a relatively high nuclear to cytoplasmic ratio. Slight irregularity in size and shape can occur, simulating dysplasia, but the chromatin is evenly distributed, finely granular, and not hyperchromatic. In the context of repair, small nucleoli and even macronucleoli may be present.

Key Features of Mature Squamous Metaplasia ( Fig. 8-14 )

• Isolated cells or loose sheets

• Distinct, densely staining cytoplasmic membrane

• Slightly dense, lightly cyanophilic cytoplasm

• Moderately transparent perinuclear zone

• Cytoplasmic projections

• Uniform, small nuclei

• Finely granular chromatin, small chromocenters.

The number of mature squamous metaplastic cells exposed for collection in cervical cytology increases with age, as the transformation zone shifts to a higher location in the endocervical canal. The most mature squamous metaplastic epithelium is indeed indistinguishable from the original mature squamous ectocervical mucosa. The majority of cervical smears from patients in reproductive age contain mature squamous metaplastic cells, recognized or unrecognized.

Mature squamous metaplastic cells tend to be isolated, less frequently occurring in loose sheets. Cells have distinct borders and are round, oval, or polygonal. Mature squamous metaplastic cells, as compared to immature metaplastic cells, have less homogeneously cyanophilic cytoplasm, and instead have a more densely staining outer zone and central perinuclear more transparent zone of cytoplasm, sometimes bearing resemblance to koilocytes. Cytoplasmic projections (“spider cells”) may be present. The cytoplasm is usually slightly dense as compared to normal ectocervical squamous cells. The nuclei are relatively small, round or oval, uniform in size, and centrally located. The chromatin is finely granular, but may form small chromocenters.

Squamous metaplasia may show atypia that overlaps with dysplastic squamous changes, in both cytology preparations and histology. These lesions can also be colposcopically abnormal, leading to common sampling of squamous metaplasia in colposcopic biopsies. To add further confusion, dysplasia often arises near the squamocolumnar junction in epithelium that is in varying degrees of squamous metaplastic maturity. When attempting to distinguish between metaplasia and dysplasia in cytologic specimens, it can be helpful to think of the cytoplasm as giving clues to the degree of cytologic maturity and nucleus as giving the most information about the degree of abnormality relating to malignant potential. Nuclei in atypical squamous metaplasia appear to be large, but only relative to the size of the cytoplasm. In absolute terms, the nuclei of squamous metaplasia are typically much smaller than dysplastic nuclei. In squamous metaplasia, nuclear hyperchromasia is generally absent. Conversely, hyperchromasia in dysplastic cells reflects an abnormality in DNA synthesis.

Key Features of Reactive and Reparative Changes ( Fig. 8-15 )

• 2-dimensional sheets of cells

• Maintained nuclear polarity

• Variation in nuclear size and shape

• Nuclear enlargement with maintained nuclear to cytoplasmic ratio

• Distinct cytoplasmic boundaries, cytoplasmic vacuolation, cytolysis

• Hyperchromatic but evenly distributed chromatin

• One or more prominent nucleoli

• Inflammatory background, neutrophils within squamous groups.

Reactive and reparative changes are usually a more acute reaction to irritation or injury than squamous metaplasia, although squamous cells in varying stages of squamous metaplasia may be affected. The most dramatic acute changes occur in response to the following: radiotherapy, systemic chemotherapy, recent surgical procedure (hysterectomy, biopsy, cone excision, ablative therapies), foreign objects (IUD, pessary, retained tampons), postpartum, uterine prolapse, and infection. Cytology specimens collected during these changes can show dramatic changes simulating high-grade malignancy. Reparative changes are generally characterized by nuclear enlargement and prominent nucleoli. Cells usually exfoliate in large, sheet-like, 2-dimensional aggregates with distinct cytoplasmic boundaries, and often with prominent streaming of the cells (sometimes referred to as like a “school of fish”). Mitotic figures may be present. Neutrophils may be admixed with the squamous groups. Rarely, abnormal singly lying cells are found. The cells have wide variation in size and shape. The cytoplasm is usually cyanophilic, but sometimes shows a dual staining reaction, and may be finely vacuolated or contain large vacuoles. Cytomegaly with abundant cytoplasm and preserved low nuclear to cytoplasmic ratios are usually observed. Cytoplasmic protrusions (“taffy pull” appendages or “spider” cells) may be present. Nuclei are round to oval, and one or more prominent nucleoli are sometimes present. As a rule, however, the chromatin is evenly distributed and not hyperchromatic. Degenerative changes may coexist with reactive and reparative changes. Degenerative changes include folded nuclear membranes, karyorrhexis, karyolysis, and pyknosis. Cytoplasmic vacuolation is a fairly consistent feature.

The most important features distinguishing reactive and reparative changes from invasive malignancy include the 2-dimensional quality of the aggregates, maintained polarity, normochromatic, evenly distributed chromatin, and macronucleoli disproportionate to the maintained low nuclear to cytoplasmic ratio. Appropriate clinical history, in conjunction with these findings, can also be helpful.

Key Features of Reactive Changes due to IUD ( Fig. 8-16 )

• Small clusters or single cells

• Cellular and nuclear size variation, multinucleation

• High nuclear to cytoplasmic ratio

• Cytoplasmic vacuolation (“bubble-gum” vacuoles)

• Nuclear hyperchromasia, but evenly distributed chromatin

• Prominent nucleoli.

Unique reactive and reparative changes have been attributed to the presence of IUDs. Both squamous and glandular changes occur. Cells may show severe cellular and nuclear pleomorphism, increased nuclear to cytoplasmic ratio, prominent nucleoli, and cytoplasmic vacuolation. Clusters of atypical cells may be present in association with the presence of an inflammatory reaction. As discussed in Chapter 9 , on glandular cervical abnormalities, IUD changes can be particularly difficult to distinguish from endometrial adenocarcinoma without the proper clinical history.