This chapter should be cited as follows: This chapter was last updated:
Lin, X, Nayar, R, et al, Glob. libr. women's med.,
(ISSN: 1756-2228) 2008; DOI 10.3843/GLOWM.10018
October 2008

Cervical carcinoma

Cytopathology

Xiaoqi Lin, MD, PhD
Department of Pathology Northwestern Memorial Hospital Northwestern University Chicago, Illinois, USA
Ritu Nayar, MD
Department of Pathology Northwestern Memorial Hospital Northwestern University Chicago, Illinois, USA
Denise D.S. De Frias, MD
Department of Pathology Northwestern Memorial Hospital Northwestern University Chicago, Illinois, USA

INTRODUCTION

Cytologic examination of the exfoliative material of the cervicovaginal area of the female genital tract has had a tremendous impact in limiting the development of invasive squamous carcinoma of the uterine cervix, therefore reducing the mortality from cervical cancer. In 1943, George Papanicolaou at Cornell University Medical College laboratories initiated a program that would become valuable as a screening test in the United States and abroad.1, 2 At that time, Papanicolaou developed a numeric classification from Class I to Class V, which indicated the absence of atypical or abnormal cells (Class I); atypical cells but no evidence of malignancy (Class II); cells suggestive of, but not conclusive for malignancy (Class III); cells strongly suggestive of malignancy (Class IV); and unquestionable evidence of malignancy (Class V). With the development and refinement of cytologic diagnosis, many believed that the Papanicolaou classification was not adequate because it did not clearly identify the nature of the lesion.3 Several modifications to the original classes were introduced and were used without the uniformity necessary for reporting abnormalities found in the uterine cervix. In 1953, Reagan and colleagues3 introduced the term dysplasia to refer to preneoplastic abnormalities of squamous cells. This classification was further subdivided into mild, moderate, and severe, indicating the variable degrees of severity of the atypical process. The advantage of this reporting system was the possibility of using it for cytologic as well as histologic samples. In 1966, Richart4 introduced the term cervical intraepithelial neoplasia (CIN), which was subdivided into grades I, II, and III, and that could also be used for grading both cytologic and histologic samples.

In 1988, a new classification, The Bethesda System (TBS), was developed by a consensus opinion at a workshop held at the National Cancer Institute in Bethesda, Maryland. The goal of TBS was to provide uniformity in the interpretation and reporting of cytologic material obtained from the cervix and vagina and thus facilitate communication between the laboratory and the clinician. TBS nomenclature requires a statement of adequacy and a descriptive diagnosis. Its nomenclature reflects the current understanding of benign, preneoplastic, and neoplastic processes of the female genital tract, with equivalents in diagnostic histopathologic terminology.5 In 1991, a second workshop was held to evaluate the impact of TBS in actual practice and to modify it where necessary. One of the major recommendations of this second meeting was that precise criteria be formulated for both the diagnostic terms and the descriptors of specimen adequacy.6 After the successful incorporation of TBS 1991 into practice, more than 90% of US laboratories used some form of TBS in reporting cervicovaginal cytology.7 With the change in practice to increased utilization of new technologies and findings from research studies in subsequent years, the Bethesda 2001 workshop was convened to re-evaluate the TBS. After a preconference Internet bulletin board process of input and a 3-day workshop that included input from pathologists, cytotechnologists, gynecologists, family practitioners, epidemiologists, attorneys, public health physicians, and patient advocates, Bethesda 2001 was developed by a consensus process.8 In the revised 2001 TBS, smears without epithelial abnormalities, including those reported previously as benign cellular changes and reactive changes are classified as no intraepithelial lesion/malignancy, epithelial neoplastic processes are reported as squamous or glandular, and other nonneoplastic findings are reported descriptively (Table 1).8 Guidelines for reporting ancillary tests, computerized screening, and educational notes and recommendations for follow-up are also included. It is left up to individual laboratories to incorporate these guidelines as best suited to their practices.

TABLE 1. Bethesda System 2001

Specimen type:

  Indicate conventional smear (Pap smear) vs. liquid-based vs. other

Specimen adequacy

  Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and any other quality indicators, e.g., partially obscuring blood, inflammation, etc.)

  Unsatisfactory for evaluation… (specify reason)

  Specimen rejected/not processed (specify reason)
  Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify reason)

General categorization (optional)

  Negative for Intraepithelial Lesion or Malignancy
  Epithelial Cell Abnormality: See Interpretation/Result (specify ‘squamous’ or ‘glandular’ as appropriate)
  Other: See Interpretation/Result (e.g., endometrial cells in a woman 40 years of age)

Automated review
  If case examined by automated device, specify device and result.

Ancillary testing

  Provide a brief description of the test methods and report the result so that it is easily understood by the clinician.

Interpretation/Result
  Negative for intraepithelial lesion or malignancy (when there is no cellular evidence of neoplasia, state this in the General Categorization above and/or in the Interpretation/Result section of the report, whether or not there are organisms or other nonneoplastic findings)

ORGANISMS:

  Trichomonas vaginalis
  Fungal organisms morphologically consistent with Candida species
  Shift in flora suggestive of bacterial vaginosis
  Bacteria morphologically consistent with Actinomyces species
  Cellular changes consistent with herpes simplex virus

Other nonneoplastic findings (Optional to report; list not inclusive):

  Reactive cellular changes associated with

  inflammation (includes typical repair)
  radiation
  intrauterine contraceptive device (IUD)

  Glandular cells status posthysterectomy
  Atrophy

Other

  Endometrial cells (in a woman > 40 years of age) (Specify if negative for squamous intraepithelial lesion)

Epithelial cell abnormalities
  Squamous cell

  Atypical squamous cells of undetermined significance (ASC-US), cannot exclude HSIL (ASC-H)
  Low-grade squamous intraepithelial lesion (LSIL) encompassing: human papilloma virus (HPV)/mild dysplasia/CIN I
  High-grade squamous intraepithelial lesion (HSIL) encompassing: moderate and severe dysplasia, CIS/CIN II and CIN III with features suspicious for invasion (if invasion is suspected)

  Squamous cell carcinoma

  GLANDULAR CELL

  Atypical

  endocervical cells (not otherwise specified [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

  Endocervical adenocarcinoma in situ
  Adenocarcinoma

  endocervical
  endometrial
  extrauterine
  NOS

  Other malignant neoplasms: (specify)
  Educational notes and suggestions (optional)

  Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included).

Solomon D, Davey D, Kurman R, et al: The 2001 Bethesda System: Terminology for reporting results of cervical cytology. JAMA 28721:2114, 2002.

 

To a certain extent, there is correlation among all of the classifications when they are compared (Table 2). TBS 2001 includes normal, inflammatory, and reactive changes as within the no intraepithelial lesion category. TBS divides squamous intraepithelial neoplasia into two groups: low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). LSIL refers to mild dysplasia and condyloma acuminata (human papilloma virus [HPV]), and HSIL refers to moderate and severe dysplasia as well as carcinoma in situ (CIS). Papanicolaou Class I correlates with benign findings and Class II indicates a benign or reactive process (negative in all classifications). Classes III, IV, and V are not as easily correlated with the other classifications. In Reagan’s classification, CIN II refers to moderate dysplasia but is considered a HSIL in the TBS, and CIN III correlates with severe dysplasia and CIS and also is classified as HSIL in the TBS.

 

TABLE 2. Reporting classifications

System

Classification

Papanicolaou

Class I

Class II

Class III, IV

Class V

Reagan

Normal

Reactive

Dysplasia

Invasive

  

Inflammatory

Carcinoma in situ

Invasive

Richart

Normal

Reactive

Cervical intraepithelial

 
  

Inflammatory

neoplasia I, II, III

 
   

Carcinoma in situ

 

Bethesda

No intraepithelial lesion (Normal, inflammatory, benign cellular changes, reactive)

Low-grade squamous intraepithelial lesion, condyloma/HPV

Invasive

   

High-grade squamous intraepithelial lesion, carcinoma in situ

 

 

HPV vaccines have been used in clinical trials.9, 10, 11, 12 Two companies manufacture two versions of HPV vaccines. Gardasil, a quadrivalent vaccine containing viral L1 capsid proteins from HPV 6, 11, 16 and 18, is made by Merck and Co and was approved by the United States Food and Drug Administration in 2006. Cervarix, a bivalent vaccine containing viral L1 capsid proteins from HPV 16 and 18, is made by GlaxoSmithKline and is awaiting for approval. The bivalent vaccine is widely available in other countries. Both vaccines have been shown to be effective in preventing CIN 2/3 and endocervical adenocarcinoma. Gardasil has been shown to be 100% efficacy against genital warts.11 The age of candidate for immunization from as young as 9 years to 26 years.13 Side-effects of quadrivalent vaccine includes injection site (pain, swelling, erythema and pruritus) and systemic effects (fever, nausea, dizziness, headache, gastroenteritis, and appendicitis) that were slightly more frequent than placebo.14 No deaths were judged to be vaccine or placebo related.14 Immunization with these vaccines will not only reduce the incidence of anogenital HPV-related diseases, but also other HPV diseases, such as oropharyngeal cancer.

SAMPLING TECHNIQUES

The accuracy of the Papanicolaou smear depends largely on obtaining an adequate cellular sample for examination. Several techniques can be used to obtain adequate samples of the uterine cervix, vagina, endocervix, and endometrial cavities.15 The adequacy depends not only, or evenly primarily, on the device that is used, but also on the technique of the individual taking the specimen.

The most commonly used and accepted techniques are an ectocervical scraping with the Ayre spatula, and endocervical brushing for endocervical canal and transformation zone sampling. The endocervical brush should be introduced gently into the canal and rotated with a gentle movement of 180 degrees. If the brush is inserted with force and with a harsh back-and-forth movement, the smear obtained will be composed mainly of blood, distorted endocervical cells showing marked pulling artifact, and muscle fibers, and the material will be inadequate for evaluation. The modified Ayre spatula with an extended tip was developed to obtain a sample from the endocervix, but it is now recognized that other instruments are superior to the spatula alone in reliably obtaining a sample that contains material from the transformation zone. More recently the endocervical broom was introduced, which can be used alone or in combination with other devices. Guidelines for taking an adequate conventional cervicovaginal smear are available.16

Liquid-based methods are currently used by many physicians to collect and preserve the material collected from the cervix and vagina. Cytyc Corporation (ThinPrep System) and Tripath imaging (SurePath)are the two vendors currently offering Food and Drug Administration (FDA)-approved liquid-based technology for cervicovaginal specimens. Liquid-based preparations may decrease preanalytic errors by fixating the sample more rapidly (preventing air drying and better preservation), decreasing artifacts and obscuring cells, such as mucus, cellular debris and red blood cells (RBCs)/white blood cells [WBCs], and homogeneously mixing the sample. There are data to suggest that utilization of liquid based technology can increase the sensitivity for detection of preneoplastic/neoplastic lesions of the cervix.17, 18, 19, 20, 21 However, regular screening remains the most effective means of cancer prevention.

Hormonal evaluation was previously performed using a gentle scrape of the upper lateral vaginal wall, but this method has now fallen in to disuse because of the availability of more sophisticated laboratory methods. However, if an abnormality is encountered on the cervicovaginal smear, in relation to the patient’s age and menstrual history, a maturation index can be issued or a comment made regarding the maturation pattern being inconsistent with age and menstrual history.

Several techniques, such as Tao brush endometrial sampler, pipelle biopsy device, glass cannula and rubber bulb have been developed to sample the endometrial cavity for cytologic evaluation.15, 22 But, none of the devices make screening for endometrial cancer feasible and no comprehensive screening program for endometrial cancer has been mounted to date.23, 24 The 2001 Bethesda Conference created a category termed "other" for reporting benign endometrial cells in women aged ≥ 40 years, with atypical endometrial cells reported as an epithelial cell abnormality.25 Recent evidence suggests that even this terminology leads to excessive investigation with little or no benefit from the detection of significant endometrial disease.26, 27 It is also noted in the Bethesda System 2001 that cervical cytology is unreliable for the detection of endometrial lesions and should not be used to evaluate causes of suspected endometrial abnormalities.25

Handling of Specimens

Proper preservation of cell detail is essential for accurate interpretation. Thus, cervical and vaginal samples should be smeared rapidly but gently on the glass slide to avoid drying the cells, which may cause marked artifacts that can preclude an adequate cytologic evaluation. Rapid immersion of the smear in 95% ethanol is the method of choice. Other alternatives for wet fixation include 100% methanol, 80% isopropanol, or denatured ethyl alcohol. When wet fixation is not possible, the most commonly used alternative method is spray fixation. The nonaerosol or pump method is preferable over the aerosol spray fixatives.

The newer thin-layer, liquid-based methods do not require smear preparation by the clinician. The cell sample is directly placed in the liquid preservative per the manufacturer’s instructions or per package insert and the thin-layer slide is prepared in the laboratory. In addition to making a thin-layer slide for cytologic assessment, the material from the vial can also be sent for ancillary testing such as HPV typing, and Chlamydia and Niesseria gonorrhea.

Cervicovaginal smears should ideally be obtained at midcycle to avoid the presence of large amounts of blood that can obscure the morphologic features of the epithelial cells. However, in some instances, bloody smears are unavoidable, and hemolysis of red blood cells can be achieved with a simple technique. The smears can be air dried for approximately 15 minutes, rehydrated in saline solution for 15–30 seconds, and fixed in 95% alcohol. Papanicolaou staining would proceed in the usual manner. Once the smears are air dried, they should be delivered at once to the cytology laboratory. The technical staff should be advised that the slides should be hydrated for 30 seconds before staining is performed, however, this technique is not useful if there is too much mucus.28 This problem is to a some degree taken care of in liquid-based collections.

Specimen Adequacy

TBS 2001 terminology for reporting results of cervical cytology made substantial changes in specimen adequacy terminology and categorization.8 TBS 2001 has two adequacy categories: “satisfactory for evaluation” and “unsatisfactory for evaluation” (see Table 1). The “satisfactory but limited by” category was eliminated. The presence or absence of endocervical/transformation zone component and any other quality indicators are provided immediately after the term “satisfactory for evaluation.” The unsatisfactory report includes reasons for the unsatisfactory designation. Criteria for adequacy evaluation on conventional and liquid based smears have been provided by the 2001 TBS guidelines.8 The evaluation of adequacy, is considered by many to be the single most important quality assurance component of TBS8 and adequacy is considered in management guidelines.29

SATISFACTORY SMEARS.

An adequate conventional smear should have a minimum of approximately 8000–12,000 well-preserved and well-visualized squamous epithelial cells. For liquid based preparations, an estimated minimum of 5000 well-visualized/well-preserved squamous epithelial cells should be seen.

While the terminology “satisfactory but limited by…” has been removed from TBS 2001, the laboratory has the option to add quality indicators (presence/absence of endocervical component, other obscuring factors, etc.) to the adequacy statement. At least 10 well-preserved endocervical or squamous metaplastic cells should be observed to report that a transformation zone component is present.

UNSATISFACTORY SMEARS.

For unsatisfactory specimens, the report should indicate whether or not the sample has been processed/evaluated by the laboratory. The wording suggested by TBS 2001 is as follows8:

  1. Specimen rejected/not processed because ___ (specimen not labeled, slide broken, etc.)
  2. Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of ___ (obscuring blood, etc.)

Additional comments/recommendations can be included in the report as deemed appropriate.

An abundance of intact or hemolyzed blood, bacteria, and RBCs can obscure the diagnostic epithelial sample, precluding adequate cytologic evaluation.30 Other causes of inadequate specimens are the presence of few epithelial cells, the presence only of endocervical cells when the ectocervix was sampled,30 and the presence of significant degenerative changes. An unsatisfactory smear is considered to be unreliable for the evaluation of epithelial cell abnormalities; however, smears that are processed and evaluated by the laboratory may provide information such as presence of blood, inflammation, organisms, etc., which may be of some clinical importance. A longitudinal study31 found that unsatisfactory cervicovaginal smears were more commonly obtained from high-risk patients, and had significantly more SIL/cancer on follow-up compared to a cohort of patients with satisfactory index smears. Both the laboratory and clinician should monitor the number of unsatisfactory smears as a quality assurance measure.

The preferred management for most women undergoing routine annual/biennial screening is a repeat smear in 12 months.32 In some studies,30 the proportion of abnormal smears has been reported to be higher in slides containing endocervical cells compared to slides lacking cells from the endocervical canal. Guidelines are available to suggest which patients may benefit from an early (within 6 months) repeat smear.32 The preferred management for most women with an unsatisfactory cervicovaginal smear result is a repeat smear, generally within a short interval of 2–4 months.32 If the unsatisfactory result is the result of obscuring inflammation and an organism is identified, specific treatment should be considered prior to repeating the smear. In cases where the smear is repeatedly unsatisfactory because of obscuring blood, inflammation, or necrosis, additional clinical evaluation such as colposcopy and/or biopsies are suggested, as clinically appropriate.32

BETHESDA 2001 SYSTEM TERMINOLOGY

General Categorization

This is an optional component of TBS 2001.8 It was designed to assist clinicians and/or their staff to triage cervical smear reports adequately and includes:

  1. No intraepithelial lesion/malignancy: encompasses the previous TBS 1991 categories of “within normal limits” and “benign cellular change/organisms” and “reactive cellular change.” These cytologic changes can be described under this category as “nonneoplastic findings.”
  2. Epithelial cell abnormalities: further specified as squamous, glandular or extrauterine/other.
  3. Other: this was added as a category for cases in which there are no cytologic abnormalities per se in the cells, however the finding may indicate some risk of a preneoplastic or neoplastic condition (e.g., endometrial cells in a woman >40 years of age).

These three general categories are mutually exclusive; thus, if several findings are present, the general categorization should be based on the most clinically significant result.8

Interpretation/Result

The unanimous opinion at the 2001 Bethesda workshop was that cervical cytology is primarily a screening test, which in certain instances may serve as a medical consultation, by providing an interpretation/result that contributes to a diagnosis.8 Because an individual patient’s final diagnosis and subsequent management, should integrate the clinical and laboratory results, in TBS 2001, the term “diagnosis” has been replaced by “interpretation” or “result.”8

Nonneoplastic Changes

Reporting of certain organisms (Trichomonas, fungi (yeast), shift in flora, Actinomyces, Herpes) is done by most laboratories; however reporting of other non-neoplastic findings is variable, and optional under TBS 2001 guidelines.8

ORGANISMS.

The vaginal flora is composed of multiple microorganisms. The most common are Lactobacillus vaginalis, Streptococcus viridans, and Staphylococcus epidermidis. They thrive in an environment in which balance is established by hormones as well as physical influences, such as intrauterine devices. Occasionally, intrauterine devices influence the balance of the vaginal flora, causing symptoms and infection with actinomyces.33

Changes caused by inflammation include metaplasia, surface reactions, such as hyperkeratosis and parakeratosis, and reparative processes.33 Frequently, the nuclei of the epithelial cells show enlargement, but the chromatin is fine, and cytoplasmic changes are variable. In other instances, degenerative changes occur.33 The background may show numerous polymorphonuclear leukocytes and debris, a large number of bacterial forms, and histiocytes. When this exudate obscures the surface of the epithelial cells, it is advisable to treat the infection and repeat the smears because dysplastic or malignant cells may be hiding beneath the white blood cells, bacteria, and debris. Nonspecific vaginitis is commonly seen in patients harboring flora such as mixed bacteria or coccobacilli. Occasionally, cells show inflammatory changes in a background of rod bacteria that appear to be Döderlein’s bacilli. These bacteria belong to a heterogeneous group of organisms, and they morphologically resemble other microorganisms that may cause inflammatory changes in the epithelial cells. The presence of some polymorphonuclear leucocytes by itself is not an indication of inflammation, reactive changes in the epithelial cells are necessary.

Bacteria.

Gardnerella vaginalis, mobilincus, and other anaerobic bacteria (bacterial vaginosis) may be found in asymptomatic women (40–50% of patients), and other women may develop leukorrhea and an inflamed vaginal mucosa (Fig. 1).33 Smears show cells covered by bacteria, so called clue cells, against a background of feathery cocobacillary forms (Fig. 1).

 Fig. 1. Clue cell showing slight enlargement of nuclei with dense cytoplasm covered by coccobacilli forms. (Papanicolaou, ×600).

Actinomyces species occur frequently in association with intrauterine device usage. Less frequently, the use of a pessary, tampons, or any type of foreign body left in place for long periods in the cervicovaginal area may elicit infection with this bacteria. Pelvic infection may occur in some patients. In Papanicolaou-stained smears, filamentous bacteria are seen, usually in clumps as well as single forms.

Chlamydia trachomatis infection is seen in cervicovaginal smears as cytoplasmic bacterial inclusions (Fig. 2). The false-positive rate is high for morphologic diagnosis, and mucus secretion in immature metaplastic cells, phagocytosed debris, and degenerative changes may be the source of erroneous diagnosis of C. trachomatis infection. Because the distinction between these entities is impossible on a morphologic basis alone, more specific microbiologic assays (polymerase chain reaction [PCR], enzyme immunoassay [EIA]) are now used for identification of chlamydia. The FDA recently approved testing for chlamydia out of the liquid-based collection devices.

Fig. 2. Squamous cell showing numerous intracytoplasmic bacterial forms consistent with Chlamydia trachomatis infection. (Papanicolaou, ×1200).

Fungi (yeast)

Candida albicans is the most common fungus found in the cervicovaginal smear (Fig. 3). Yeast forms as well as pseudohyphae are commonly encountered. Diabetes, pregnancy, birth control pills, and antibiotic use may change the vaginal flora, and predispose patients to infection by this fungal agent. Fewer than 10% of mycotic vaginal infections are caused by another Candida species, Torulopsis glabrata, which is morphologically similar to the yeast form of C. albicans.

  Fig. 3. Pseudohyphae of Candida albicans. (Papanicolaou, ×400).

Flagellates

Trichomonas vaginalis infection may cause changes that range from minimal to severe. Fifty percent of infected women are asymptomatic.33 The classic signs of inflammation are present in the epithelial cells and also in the background of the smears that contain variable amounts of debris and WBCs. Small perinuclear halos may be seen in the squamous cells, but this finding is not pathognomonic of this infectious process. The organisms are seen as pear-shaped structures with a punctate, almond-shaped nucleus. Flagella rarely are seen in smears prepared with the Papanicolaou technique. Without the presence of nuclei, a diagnosis of T. vaginalis cannot be made because fragments of cytoplasm of the intermediate squamous cells may resemble these flagellates. Leptothrix (Leptotrichia buccalis) are filamentous bacteria that frequently are associated with T. vaginalis (70–80% of cases) (Fig. 4). Certain forms of Döderlein’s bacilli are morphologically identical to Leptotrichia, and only by culture can they be identified adequately.

   Fig. 4. Trichomonas vaginalis with squamous cell change and filamentous bacteria (Leptothrix). (Papanicolaou, ×600).

 

 

 

 

Viruses.

The most common viral infections encountered in cervicovaginal smears are caused by papillomaviruses and herpes genitalis (Fig. 5). Papillomaviruses are the principle etiology of cervical neoplasia. Herpes genitalis infection occurs primarily by sexual transmission. Clinically symptomatic eruptions may occur. Usually, they are related to stress, the menstrual period, or unrelated diseases. Cytologically, the epithelial cells show multinucleation, the chromatin assumes a ground-glass appearance, and intranuclear inclusions may appear in some cells.

   Fig. 5. Herpes simplex virus infection (Papanicolaou, ×600)

 

 

 

 

 

Over the years, there have been several reports in the literature suggesting that microorganisms that induce changes in the cervical epithelium, may be a potentiating cause for the development of cervical neoplasia. Trichomonas vaginalis,34 Chlamydia trachomatis,35 and herpes genitalis36, 37 cause inflammatory changes that not infrequently associated with SIL and may produce fertile ground for the development of preneoplastic and neoplastic squamous lesions. Immunosuppression may be a setting for viral infection, such as HPV and herpes simplex, and also for the development of neoplasia.38 However, it was not until the 1970s that the tools of modern molecular biology were applied to the molecular characterization of the papillomavirus family that HPV,39 with more than 100 molecular types, emerged as the major player in the etiology of cervical neoplasia.

BENIGN/REACTIVE CHANGES.

Inflammation and Regenerative Changes.

A common finding in cervicovaginal smears is reparative change as a result of a variety of processes that may involve the glandular, metaplastic, and squamous epithelium.40 These changes are seen after damage to the mucosa and after radiation, chemotherapy, infections, or any condition that alters the tissue integrity. When a reparative process is present, cellular samples display monolayer sheets of elongated, large, immature cells with a regular arrangement and conserved polarity. The nuclei are enlarged and contain pale, powdery and evenly distributed chromatin and nucleoli. The cytoplasm is delicate and possibly thickened with possibly variable staining quality and ill-defined borders. Isolated atypical cells of a reparative nature are rare (Fig. 6)41, 42. Inflammatory changes in epithelial cells are differentiated from dysplasias and carcinomas by the relative low content of chromatin, symmetrical nuclear outline, and low nuclear–cytoplasmic ratio. When inflammatory/reparaties cause epithelial atypia, in either squamous or endocervical cells, the changes maybe classified as atypical squamous cells (ASC) or atypical glandular cells (ACG). Follow-up is needed in these cases to make sure the atypia has regressed.

    Fig. 6. Reparative process with inflammatory changes. The cells are arranged in monolayers and contain enlarged nucleoli. (Papanicolaou, ×400).

Differences exist between these proliferating cells and invasive squamous carcinoma, such as the absence of single atypical cells, macronucleoli, a low nuclear–cytoplasmic ratio, and the absence of tumor diathesis (Fig. 6), and are important for the differentiation of repair from nonkeratinized squamous carcinoma and, rarely, from sarcomas.40

Therapy Changes.

Several modalities of therapy affect malignant and benign cells, the latter at the vicinity or at a distance from the malignant neoplasm. The types of therapy include chemotherapy, immunotherapy, radiation therapy, and mechanical therapy.43 Therapy of any modality may cause changes in benign cells that can resemble malignant changes.

By examining cervicovaginal smears, short- and long-term radiation changes can be observed.41, 44 In benign cells, short-term changes usually are found within 6 months of irradiation. These changes consist of nuclear enlargement showing degenerative changes (nuclear pallor, wrinkling or smudging of condensation of chromatin), prominent single or multiple nucleoli, abnormal mitosis, and multinucleation. At the cytoplasmic level, there are varying degrees of vacuolization and/or polychromatic staining. Cellular enlargement occurs, and a low nuclear–cytoplasmic ratio is maintained. This feature helps the cytopathologist to recognize the affected cells as benign.41, 42 Short-term changes can also be encountered in malignant cells shortly after treatment. However, with effective treatment, they regress approximately 1 month after the completion of therapy because of their exquisite sensitivity to radiation.44 During the first month, an exudate contains leukocytes and debris resulting from local necrosis. Mononucleated and multinucleated histiocytes are often present. Malignant cells that show radiation changes 6–8 weeks after treatment indicate persistence of the neoplasm. After this time, the presence of malignant cells without radiation changes indicates recurrence.

The features seen with long-term radiation changes are reflected in cytologic smears by aberrant cells from the basal layers, cellular enlargement, and atrophy. These changes can persist for several years, and vary from patient to patient. Under light microscopy, cytoplasmic vacuolization starts at the parabasal layers, and engulfment of polymorphonuclear leukocytes within some of the vacuoles are seen. Some of the cells acquire an abnormal staining quality referred to as amphophilia. The most important diagnostic characteristic change is significant enlargement of the epithelial cells, with otherwise normal features. Giant-cell histiocytes, and reparative changes,45 are usually encountered. The effectiveness of radiation can be monitored by the assessment of several cellular features in cervico-vaginal smears.44, 46 The cytopathologist should observe the postradiation smear for increased cellular maturation, which is a warning sign for the development of de novo intraepithelial neoplasia. Equally important is the detection of tumor diathesis, which may indicate persistence or recurrence of the tumor. True tumor diathesis has necrotic cells with nuclear and cytoplasmic fragmentation, hemolyzed blood, and debris; the mere presence of a “grungy” background is not sufficient to qualify.

Systemic chemotherapy may affect cells exfoliated from the cervicovaginal area. Cellular changes bear some similarities to changes caused by ionizing radiation.41 Occasionally, the cells look highly abnormal, except for the maintenance of the amount of cytoplasm and the absence of mitotic figures.

Immunodeficiency, including those caused by immunosuppressive therapies, increases in the number of neoplasms at several body sites, including the vulva and cervix.47 The incidence of cervical neoplasms has been reported to reach a 14-fold increase compared with the incidence in the nonimmunosuppressed population. It is advisable to have a baseline cervicovaginal smear to monitor the development of neoplasms during immunosuppressive therapy.43

Electrocautery of the uterine cervix produces coagulative cellular necrosis and significant inflammatory response represented by polymorphonuclear leukocytes and lymphocytes.41, 43 These changes may mimic a tumor diathesis background. In addition, cervicovaginal smears may harbor atypical cells after electrocautery treatment for benign processes. Because these cells may persist for several weeks,48 a waiting period of 1–3 months should be allowed to permit the necrosis and healing process to occur.41, 43, 48 These changes are not limited to the squamous epithelium; atypia of the endocervical cells is also encountered.43 For these reasons, a history of electrocautery, as well as the date of therapy for neoplastic and nonneoplastic processes, should be disclosed to the pathologist to avoid misinterpretation of cervicovaginal smears. Laser therapy has been used for years to eradicate dysplasia and CIS of the uterine cervix.49, 50 The carbon dioxide laser produces a localized intense heat that inactivates cellular components, interrupting the replication process at the cellular level.50 Laser therapy has several advantages over cryotherapy with respect to cervicovaginal smears. Because of the almost-complete absence of bleeding and necrosis, as well as the faster healing process, the inherent difficulties (atypical cells and inflammatory background) in cryotherapy are short-lived with laser treatment, and the cervicovaginal smears return to normal quickly.49, 50 The regenerative process takes place from the epithelial edges of the ulcer that is produced by the laser and fills the gap within 2 weeks. In spite of a positive Schiller’s test result, the cells desquamated from this site are easily recognized as benign.49 After approximately 6 weeks, this test result becomes negative as the epithelium matures in the newly re-epithelialized site.

Benign Surface Reactions of Squamous Epithelium.

Hyperkeratosis.

Under the influence of a variety of stimuli, the surface of the squamous epithelium may develop a granular layer and several layers of anucleated cells as a protective mechanism. This phenomenon is referred to as hyperkeratosis (Fig. 7A). Clinically, this condition is seen as an area of leukoplakia; cytologically, the smears contain single or groups of anucleated squames (Fig. 7A).42, 51 The cells derived from the granular layer show keratohyaline cytoplasmic granules (Fig. 7B).

Fig. 7A. Histologic section of squamous mucosa showing a granular layer and hyperkeratotic layers (Hematoxylin and eosin, ×360). Inset. Clusters of anucleated squames from the epithelial surface. (Papanicolaou, ×650).

  Fig. 7B. Superficial cells showing keratohyaline granules and isolated anucleated squames. (Papanicolaou, ×600).

Thick patches of those cells with irregular outlines seen consistently throughout the smear are of diagnostic importance. Keratohyaline granules and a few anucleated squames are normally seen at the height of estrogenic stimulation at midcycle and have no clinical significance. However, the presence of many of these cells, associated with patches of anucleated squames, indicates hyperkeratosis and presence of a granular layer in the cervical mucosa. Occasional anucleated cells on the edge of the slides or in the specimen should not be mentioned in the diagnosis because they probably represent contamination from the patient’s vulva or from the fingers of the person handling the slides. It is thus advisable to report only cells with keratohyaline granules associated with anucleated squames that are intermixed with other normal components of the smears. Extensive hyperkeratosis, when appropriately identified, may be associated with underlying abnormal epithelium in 29% of patients.51

Parakeratosis.

Parakeratosis is another type of surface reaction that is characterized by a proliferation of layers of small nucleated squamous cells with pyknotic nuclei. In smears, miniature keratinized squamous cells are seen, either singly or in sheets (Fig. 8A). The nuclei are small and either round or oval. The nuclear–cytoplasmic ratio is low. Parakeratotic cells without nuclear abnormalities can be seen in a variety of situations, such as inflammation, pessary use, and other processes that are irritative to the cervical mucosa. When they are enlarged and irregular, they are classified as atypical squamous cells or atypical parakeratosis (see Fig. 8B).40 Atypical parakeratosis correlates with the presence of an underlying SIL or invasive carcinoma in approximately 40% of patients (Fig. 8C, LSIL).52 Once a diagnosis of atypical parakeratosis is made, the smears should be repeated with a forceful scrape to obtain cells of deep layers. Some authors45 advise that two smears should be taken, the first to remove the parakeratotic cells and the second to obtain deeper cells, which may be abnormal.

 Fig. 8. A: Parakeratotic cells without atypia. B: Atypical parakeratotic cells. The nucleus of the parakeratotic cell is enlarged and opaque. C: LSIL. The dysplastic cell has a marked enlarged atypical nucleus, with coarse granular chromatin. (Papanicolaou, ×600).

Intrauterine Device Effect.

The presence of an intrauterine device in the endocervical and endometrial cavity causes an inflammatory response and subsequent irregular desquamation and shedding of these epithelial cells from these areas may mimic glandular or squamous neoplasm. Squamous metaplasia showing variable degrees of maturation is frequently seen in ectocervical-endocervical smears. The immature metaplastic cells may maintain the mucus-producing ability inherent to endocervical cells because they have a similar histogenesis. This feature mimics the appearance of atypical glandular cells, and may be difficult for the cytologist to identify. Immature metaplasia in single cells with nuclear enlargement only, may mimic high-grade squamous dysplasia. Atypical glandular cells of endocervical43, 53 or endometrial origin also may be encountered.41, 54 They can be recognized as reactive or benign if clinical information about intrauterine device use is disclosed, avoiding false-positive results.54 A less common cell type, referred to as indeterminate, has been postulated as being of endometrial origin.55 Few of these cells are seen in a cervicovaginal smear, and they have a variety of features that distinguish them from CIS, such as multinucleation, presence of enlarged nucleoli, and absence of other atypical cells in the smear showing a spectrum of dysplastic changes.43 After removal of the device, these cells disappear from cervicovaginal smears after 1 month to 1 year.55

Microglandular Hyperplasia.

Microglandular hyperplasia has been described in patients taking oral contraceptives, in pregnancy, and postpartum. Therefore, it was considered a reflection of the action of progestrogenic stimulation on the endocervical epithelium. However, cases have also been seen in patients with no history of hormonal intake and occasionally in patients with hyperestrogenism or exogenous estrogenic therapy. It is not considered to be a preneoplastic change. Florid microglandular hyperplasia in histologic sections may architecturally resemble clear cell adenocarcinoma.56 Lack of stromal invasion, rare mitosis, and intracellular glycogen excludes the diagnosis of malignancy. Microglandular hyperplasia is a common finding in cervicovaginal smears. The classic appearance of the smears is of numerous dispersed rounded endocervical cells (Fig. 9). Some show a pseudokeratinized appearance caused by ischemic necrosis; hence, the misnomer pseudoparakeratosis was used in the past. Cytologically, some cases maybe mistaken for atypical squamous or atypical glandular cells.

 Fig. 9. Isolated rounded endocervical cells from a patient with microglandular hyperplasia. (Papanicolaou, ×1200).

Squamous Epithelial Lesions

DEVELOPMENT AND CLASSIFICATION.

At the beginning of the last century, the concept that intraepithelial neoplasia antedated the development of invasive squamous carcinomas was postulated by Rubin.4, 57 With increased sophistication in the field of cytopathology, it was found that analysis of cellular samples could provide a precise diagnosis that could be correlated with histologic samples. Additional work and increased awareness of the value of cytology led pathologists and gynecologists to realize that even more incipient lesions, such as dysplasias of varying degrees, could be assessed correctly in smears and supported by colposcopic and histologic studies. The terms dysplasia and CIS, as well as CIN, were introduced to unify the cytologic and corresponding histologic assessment of squamous lesions of the uterine cervix.3, 4, 58, 59, 60 The concept was that intraepithelial lesions represent a continuum, and in some cases, lesions of variable degrees can be found in the same cervix, and can be detected by cytologic and histologic methods. Dysplasia refers to a lesion that shows abnormal cells replacing a portion of the epithelial thickness.

To understand the histogenesis of intraepithelial neoplasia and invasive carcinoma, knowledge of some basic morphologic concepts is necessary (see Fig. 10). The endocervical canal is composed of tall columnar ciliated and nonciliated and mucus-producing cells. Normally, the endocervical epithelium is attached directly to the stroma without a basal layer. Under the mucosa, there are isolated immature totipotential cells that are the source of development of the endocervical cells. Under a variety of hormonal or physical stimuli, these germinative cells may proliferate, giving rise to reserve-cell hyperplasia (Fig. 11). Reserve-cell hyperplasia may develop in young patients as a result of physical injury and in postmenopausal patients because of hormonal stimuli, or from hormones produced by the adrenal gland that are later metabolized to estrone.61 The cells desquamated from reserve-cell hyperplasia have small nuclei, finely granular chromatin, and small amounts of cytoplasm. Caution should be taken not to overdiagnose them as CIS in cytologic and histologic samples. They have a monomorphic appearance, do not display hyperchromasia, and usually are seen in organized small clusters (see Fig. 11). A neoplastic process may develop in those cells as CIS of the small cell type (Fig. 12) and, later, as small cell invasive carcinoma (see Fig. 10 and Fig. 13). Small cell undifferentiated squamous CIS is seen as small cells with hyperchromatic nuclei with limited amounts of cytoplasm. To a certain extent, the features of invasive small cell carcinoma are similar to those of the intraepithelial form. However, tumor diathesis, that is necrosis, hemolyzed blood, and cellular debris, and enlarged nucleoli frequently are associated with the invasive type.

Fig. 10. Morphogenesis of carcinoma of the uterine cervix. (Wied GL, Keebler CM, Koss LG, Reagan JW (eds): Compendium of Diagnostic Cytology, 6th edn. Chicago, Tutorials of Cytology, 1990: 108. )

Fig. 11. Histologic section showing reserve-cell hyperplasia and squamous metaplasia of the endocervix (Hematoxylin and eosin, ×260). Inset. Reserve-cell hyperplasia and squamous metaplasia of the endocervix. The cells are small and monomorphic, with finely distributed chromatin. (Papanicolaou, ×360).

  Fig. 12. Syncytial arrangement of a sample from a patient with small cell carcinoma in situ. (Papanicolaou, ×600).

Fig. 13. A group of malignant cells from a patient with small-cell invasive carcinoma. The chromatin is coarse, and nucleoli are evident. (Papanicolaou, ×1200).

By a process of maturation, the reserve cells may acquire squamous features, and squamous metaplasia will replace the original glandular epithelium (see Fig. 10 and Fig. 11). The stimuli inducing those changes can be physical, inflammatory, or hormonal.45, 61 Squamous metaplasia is a benign process found frequently in smears. As a benign component, it should be recognized for adequacy of sampling because its presence indicates adequate sampling of the transformation zone (Fig. 14). However, under the influence of an unknown stimulus, preneoplastic and neoplastic changes may take place in a minority of cases. In those instances, metaplastic dysplasia of varying degrees (Fig. 15 and Fig. 16), nonkeratinizing dysplasia or CIS (see Fig. 16 and Fig. 17), or nonkeratinized invasive squamous carcinoma may develop.59, 60

  Fig. 14. Squamous metaplastic cells surrounding a superficial squamous cell. (Papanicolaou, ×600).

 Fig. 15. Metaplastic dysplastic cells and a superficial squamous cell. The cytoplasm is well defined, the nucleocytoplasmic ratio is increased, and nuclear pleomorphism is noticeable. (Papanicolaou, ×600).

Fig. 16. Key to cells derived from normal epithelia, benign proliferative reactions, and preinvasive reaction of the uterine cervix. (Wied GL, Keebler CM, Koss LG, Reagan JW (eds): Compendium of Diagnostic Cytology, 6th edn. Chicago, Tutorials of Cytology, 1990: 35, 36. )

 Fig. 17. Nonkeratinized dysplastic cells showing abnormal nuclei and delicate cytoplasm. On the lower left side, a monolayer of normal endocervical cells is seen. (Papanicolaou, × 900).

By sampling the surface of the abnormal mucosa, it is possible to evaluate the process of maturation from the basal to the uppermost layers.45 The stimulus that induces maturation is related directly to the degree of differentiation of the abnormal basal layers. The less differentiated cells will respond poorly to stimuli inducing maturation, and will emerge to the surface and a higher degree of abnormality will be seen when the surface is sampled.

CURRENT TERMINOLOGY FOR SQUAMOUS INTRAEPITHELIAL LESIONS.

The 1988 TBS,5 introduced a two-tiered terminology, dividing the spectrum of squamous epithelial abnormalities into LSIL and HSIL lesions. Low-grade lesions encompass the cellular changes associated with the cytopathic effect of HPV, also referred to as koilocytotic atypia, and mild dysplasia/CIN I. High-grade lesions encompass moderate dysplasia, severe dysplasia, and CIS/CIN II/III. TBS divides the spectrum of SIL cytologic abnormalities into two categories for the following reasons: (1) studies have reported poor interobserver and intraobserver reproducibility with conventional three- and four-grade classification systems, (2) in the United States, the difference between moderate/severe dysplasia and CIS is not relevant to patient management, (3) data from natural history studies comparing low- and high-grade lesions imply differences between these two lesions, although the biologic behavior of any individual abnormality, whether high or low grade, cannot be predicted.6

The rationale of introducing the term squamous intra-epithelial lesion (SIL) to replace dysplasia/CIN, was based on observations that most mild dysplasia/CIN regress, approximately half of the cases of moderate dysplasia/CIN do not progress, and as a group, high-grade lesions are more likely to progress than low-grade lesions, but the behavior in an individual patient is unpredictable.6 The term lesion rather than neoplasia was chosen to convey the uncertain biologic potential in any individual patient.6

The dichotomous classification of SIL (LSIL/HSIL) was retained in TBS 2001, because it seems to be supported by current virologic, molecular, and clinical evidence that LSIL is generally a transient infection with HPV, and high-grade SIL is more often associated with persistence of HPV and higher incidence of progression.8 In addition, data from the ASCUS/LSIL Triage Study (ALTS), trial has further supported the two-tier terminology by demonstrating that (1) LSIL/HSIL is a fairly reproducible breakpoint, (2) confirmed that subdividing HSIL into moderate and severe dysplasia is not reproducible, and (3) HPV cytopathic effect cannot be reliably separated from mild dysplasia/CIN I.6, 8

The 2001 Bethesda System8 suggests reporting squamous epithelial abnormalities as follows: atypical squamous cells of undetermined significance, (ASC-US); atypical squamous cells/rule out HSIL, (ASC-H); LSIL; and HSIL. Recently, some authors suggested to add one more category, LSIL, cannot exclude HSIL.62, 63

Approximately 50 million Pap smears are performed each year in the United States,64 of which approximately 7% (3.5 million) are reported as abnormal.65 Among these, approximately 3 million women have LSIL or ASC smears.66

Atypical Squamous Cells.

TERMINOLOGY.

Atypical squamous cells of undetermined significance (ASCUS) was introduced into TBS terminology to represent squamous cell abnormalities that are more marked than those attributable to reactive changes but that quantitatively or qualitatively fall short of a definitive diagnosis of SIL.6 The cellular changes in this category may represent a reactive/reparative/benign change or a more serious lesion; however, they cannot be unequivocally classified by the pathologist, and are thus interpreted to be of undetermined significance. The cellular changes include nuclear enlargement to two and half to three times that of a normal intermediate squamous cell nucleus, a slightly increased nuclear–cytoplasmic ratio; with possible variation in nuclear size and shape, and binucleation. The nuclei may also show mild hyperchromasia, but the chromatin remains evenly distributed without granularity and nuclear outlines usually are smooth and regular.6 TBS 1991 encouraged pathologists to qualify ASCUS as to whether a reactive process or SIL was favored,6 however in actual practice, a large number of these cases were reported as atypical squamous cells of undetermined significance, not otherwise specified (ASCUS-NOS).8

A diagnosis of ASCUS can be caused by a variety of etiologic factors6 such as inflammation and repair, and both qualitative (poorly fixed smear) and quantitative (few/rare abnormal cells) reasons may contribute to the pathologist making this diagnosis. Keratotic cells shed singly or in three-dimensional clusters that demonstrate cellular pleomorphism (caudate or elongate shapes) and/or increased nuclear size or chromasia should be categorized as ASCUS (atypical parakeratosis) or SIL depending on the degree of the cellular/nuclear abnormalities.6

Despite efforts to provide specific criteria for the diagnosis of ASCUS, this diagnosis is poorly reproducible.8 In order for laboratories not to overuse this term, it is suggested that the frequency of ASCUS diagnoses should not exceed two to three times the rate of SIL in the same laboratory6 or the ASCUS: SIL ratio should not be greater than 2–3:1. As an example, if the frequency of SIL in a laboratory practice is 1%, the frequency of ASCUS should be less than or equal to 3%.6

TBS 2001 retained this equivocal category of reporting atypical squamous cells because approximately 10–20% of women with ASCUS may have underlying CIN II/III, and 1 in 1000 may have invasive carcinoma.66 However, the TBS 2001 classification differs in several ways with regard to reporting equivocal squamous epithelial abnormalities. ASC are now qualified either as being of “undetermined significance” (ASC-US) or as “cannot exclude HSIL” (ASC-H). The “undetermined significance” qualifier was retained to emphasize that some cases of ASCUS maybe associated with an underlying HSIL. Additionally, ASCUS-favor reactive was eliminated; all ASC are considered now to be suggestive of SIL.8

Atypical Squamous Cells of Undetermined Significance.

In the vast majority, 90–95%, of cases, ASC involves nuclear enlargement in squamous cells with mature, superficial/intermediate-type cytoplasm8, 66 (Figs. 18A, 19, 20, and 21). The differential diagnosis is most often between a benign change in reaction to a stimulus versus a LSIL.8 This is the category of ASC-US that is now referred to as ASCUS in TBS 2001. Cells with cytologic features diagnostic of HPV cytopathic effect, that it, a well-defined, clear, perinuclear cavity with a peripheral rim of thickened cytoplasm as well as required nuclear alterations, are classified as LSIL6 (Fig 18B). Cells with features that are suggestive but not diagnostic of HPV cytopathic effects, are included in the ASC-US category. Cytoplasmic vacuolization alone, without any nuclear atypia, is considered to be a benign cellular change and should not be classified as LSIL or ASCUS.6

  Fig. 18. A. Sign of human papilloma virus (HPV) infection: mild koilocytosis (koilocytes without nuclear atypia). This may be interpreted as atypical squamous cells of undetermined significance (ASCUS). (Papanicolaou, ×600). B. Koilocytes showing large defined perinuclear halos, binucleation, and slight nuclear atypia, with smudged chromatin. This would be classified as low-grade squamous intraepithelial lesions (LSIL). (Papanicolaou, ×600).

Fig. 19. Sign of human papilloma virus (HPV) infection: cells with cracking of cytoplasm/keratohyaline-like granules. (Papanicolaou, × 900).

 

Fig. 20. Sign of human papilloma virus (HPV) infection: cells with spindled nuclei. (Papanicolaou, ×900).

 Fig. 21. Atypical squamous cell of undetermined significance (ASCUS): a sheet of intermediate squamous cells with mild nuclear enlargement and fine chromatin. Differential diagnosis is low-grade squamous intraepithelial lesion (LSIL) and reactive change. (Papanicolaou, ×600).

Marked cellular changes involving tissue fragments, or sheets of immature squamous cells, so-called atypical repair, also are included in the ASC category, but present a different cytologic picture. In typical repair, cells occur primarily in monolayer sheets and syncytia and contain prominent nucleoli. However, nuclear piling, significant anisonucleosis, and irregularities in chromatin distribution that exceed changes seen in typical repair were considered to be ASCUS in TBS 1991.6 The differential diagnosis is between an exuberant reparative process versus invasive carcinoma, however, atypical repair lacks both a tumor diathesis and isolated abnormal cells seen in squamous cell carcinoma.6 A variety of reactive cellular changes, such as degeneration, autolysis, etc., may be seen in an atrophic smear and occasionally they can mimic HSIL or squamous carcinoma. A diagnosis of ASCUS associated with atrophy should be considered if cells demonstrate: nuclear enlargement (at least two times normal), significant hyperchromasia, nuclear chromatin or membrane abnormalities, or marked pleomorphism in the form of tadpole or spindle cells.6

The ALTS, a multicenter trial sponsored by the National Cancer Institute, showed that HPV DNA testing for high-risk types is a viable option for management of women with ASC-US, and has a greater sensitivity to detect CIN III or above and specificity comparable to a single additional cytologic diagnosis of ASC-US or above.66 The American Society for Colposcopy and Cervical Pathology (ASCCP) has published consensus guidelines for the management of women with cervical cytologic abnormalities, based on the TBS 2001 terminology.29 The guidelines support DNA testing for high-risk types of HPV for an ASC-US cytologic diagnosis. Women who test positive for HPV DNA should be referred to colposcopy and those who test negative for high-risk HPV-DNA can be followed up with repeat cytologic testing at 12 months. If no lesion is found at colposcopy in a high-risk HPV–positive woman, follow-up cytology at 6 and 12 months or HPV-DNA testing at 12 months are suggested options.29 Reflex test orders from the clinician at the time of obtaining the sample allow the laboratory to send the same specimen collected for the cervical smear for HPV testing, avoiding a return visit, and 40–60% women will be spared a colposcopic examination. For ASCUS in postmenopausal women, a course of intravaginal estrogen followed by a repeat cytologic test obtained approximately 1 week after completion of the estrogen test is acceptable if the smear is atrophic and there is no contraindication to using intravaginal estrogen.29 Immunosuppressed women with ASCUS should be referred to colposcopy and pregnant women should be managed in the same manner as nonpregnant patients with ASC-US.29

Atypical Squamous Cells/Rule Out High-Grade Squamous Intraepithelial Lesions.

In a smaller number of ASC cases, 5–10% of all ASC, the atypical changes occur in less mature squamous metaplastic cells, previously called atypical metaplasia.8 This category has been reclassified as ASCH or atypical squamous cells/rule out HSIL, in TBS 2001, and reflects a mixture of HSIL and its imitators.8 Nuclear enlargement approximates one and a half to two times the area of a mature squamous metaplastic nucleus, or three times the area of a normal squamous intermediate-cell nucleus (Fig. 22). In these cases, the differential diagnosis is between immature squamous metaplasia and a HSIL and LSIL is not a consideration.6

 Fig. 22. Atypical squamous cell, rule out high-grade squamous intraepithelial lesion (ASCH): a single cell with high N/C ratio and nuclear hyperchromasia and irregularity. Differential diagnosis is high-grade intraepithelial lesion (HSIL) and immature squamous metaplasia. (Papanicolaou, ×600).

While there is interpreter variability in making the diagnosis of ASC, even among expert cytopathologists,67 studies suggest that ASC-H has a positive predictive value for histologic CIN II/III that is intermediate between ASC-US and HSIL.8 A woman with ASC-US has a 5–17% chance of having CIN II/III confirmed by biopsy, while CIN II/III is identified in 24–94% of those with ASC-H.29, 66, 68 The recommended management for women with ASC-H obtained using conventional or liquid-based cytology is referral for colposcopic evaluation.29 There is no role for HPV testing or repeat cytology. If no lesion is found at colposcopic, a review of cytology, colposcopy, and histology results should be performed. If the cytologic diagnosis of ASCH is upheld and no revised interpretations are found, cytologic follow-up at 6 and 12 months or HPV-DNA testing at 12 months are acceptable.29

Low-Grade Squamous Intraepithelial Lesion.

On histology, mild dysplasia is diagnosed when the upper two thirds of the abnormal squamous epithelium shows an adequate degree of differentiation. In many institutions, koilocytic or HPV change maybe reported on biopsy. Corresponding cytologic samples in both cases show abnormal cells with mature or superficial-type cytoplasm.6 In LSIL, nuclear enlargement is at least three times the size of a normal intermediate cell nucleus (Fig. 23).6 Although the nucleus is hyperchromatic, the chromatin is distributed uniformly or it may appear degenerated and smudged if associated with cytopathic changes induced by HPV (Fig. 18B). Cells with HPV cytopathic effect (koilocytes), are also interpreted as LSIL on cytologic smears (Fig. 18B). In these cases, the nucleus may not be enlarged, but is usually hyperchromatic and “wrinkled.”

 Fig. 23. LSIL. The nuclei of some cells are more than 3 times larger than the size of normal intermediate cell nucleus and hyperchromasia with smooth or irregular nuclear membranes. (Papanicolaou, ×600).

 

 

 

While most LSIL, especially in young women regress/are self- limited HPV infections,68, 69 approximately 15% to 30% of women with LSIL on cervical cytology will have a CIN II/III on a subsequent cervical biopsy. The ALTS trial has shown that HPV DNA testing is not useful in triaging women with LSIL because 83% were positive for HPV.66 ASCCP guidelines29 currently recommend colposcopy as the preferred management for women with LSIL. Subsequent management will depend on whether a lesion is identified, whether the colposcopy is satisfactory, and whether the woman is pregnant. Routine diagnostic excisional procedures such as loop electrosurgical excision procedure (LEEP) are discouraged for initial management of women with LSIL in the absence of biopsy-confirmed CIN. Special recommendations for postmenopausal women, adolescents, and pregnant women are also outlined in the ASCCP consensus publication.29

High-Grade Squamous Intraepithelial Lesion.

In the TBS terminology, HSIL encompasses moderate and severe dysplasia and CIS. In moderate dysplasia, the morphologic appearance of the upper one third of the epithelium is relatively preserved in contrast with abnormal changes of the underlying layers. Cytologically moderate dysplasia shows cells with less cytoplasm, larger nuclei, and occasionally with asymmetrical nuclear outlines. The chromatin is increased and granular. In severe dysplasia or CIS, histology shows an epithelium that has immature cells throughout its thickness. These changes are seen in cytologic smears as immature cells, with scant cytoplasm and a high nuclear–cytoplasmic ratio (Fig. 24). The abnormal cells can be seen singly or in crowded, dark sheets/groups. At times it may not be possible to exclude the possibility of invasive carcinoma and in such cases, the terminology for reporting this finding is “HSIL, with features suspicious for invasion.” The incidence of CIN II/III is estimated to be approximately four times that of invasive carcinoma. All patients with a HSIL cytologic diagnosis should be referred for an immediate colposcopic evaluation.29

 Fig. 24. High-grade squamous intraepithelial lesion (HSIL) cells showing moderate-to-severe dysplasia. (Papanicolaou, ×600).

Borderline Squamous Intraepithelial Lesion.

For practical purposes, the cells from mild dysplasia/HPV cytopathic effect show abundant cytoplasm with enlarged or hyperchromatic, wrinkled nuclei (Fig. 18B). As the lesion progresses from moderate-to-severe dysplasia (Fig. 24), the amount of cytoplasm decreases as the nucleus increases in size, increasing the nuclear–cytoplasmic ratio.

At times there maybe difficulty in categorizing cells with intermediate features into low or high grade. Although occasional borderline cases occur, most of these can be classified as either LSIL or HSIL. Features that favor a high-grade lesion include increased numbers of abnormal cells, higher nuclear/cytoplasmic ratios, greater irregularities in the outline of the nuclear envelope, coarsening of nuclear chromatin, and chromatin clumping.6 Cell size, overall is smaller in HSIL as compared to LSIL. The appearance of the cytoplasm is usually different in LSIL and HSIL cases. LSIL typically involves squamous cells with mature, intermediate, or superficial-type cytoplasm with well-defined polygonal cell borders, whereas cells from a HSIL have a more immature type of cytoplasm, that can be lacy and delicate or dense/metaplastic, with rounded cell borders.6 When it is not possible to grade a SIL as low or high grade, a diagnosis of “SIL, grade cannot be determined” or “LSIL with few cells suggestive of HSIL” can be rendered on the cervical smear. These patients should under colposcopy/biopsy.6

Progression and Regression of Squamous Intraepithelial Neoplasia.

Spontaneous regression of ASCUS occurs with great frequency (68%),70 LSIL regresses in 47% or more,45, 69, 70 and HSIL regresses in approximately 35–40% of cases.58, 70 Most severely abnormal lesions are destroyed by a variety of modalities of treatment. Only a tiny fraction of all dysplasias, left untreated, would progress to invasive cancer, but the risk increases with increasing grade of the lesion (7–10% for ASCUS, 15–21% for LSIL, and 23% for HSIL).70, 71 

Intraepithelial Neoplasia in Pregnancy.

During the first trimester of pregnancy, dysplastic cells are smaller than in nonpregnant patients, thus giving the false impression of a higher grade of abnormality.72, 73 In the late stages of pregnancy, the size of the cells is comparable to the nongravid population. The biologic growth rate is similar to that of the general population. Most cases of LSIL regress or remain stable during pregnancy.74 All cases of HSIL diagnosed antepartum persisted in the postpartum period, and 11% of patients with antepartum HSILs are found to have invasion noted 2–5 years after diagnosis of SIL in the antepartum. The possibility of undetected intraepithelial lesions may be higher than in nonpregnant patients because of a larger number of unsatisfactory smears.75

 

PERITONEAL WASHINGS

Peritoneal washing cytology performed during surgery or second-look laparoscopy for carcinoma is used by gynecologic oncologists for the management of patients with gynecologic malignancies, specifically ovarian and endometrial carcinomas.76, 77 It can also be used in cervical carcinoma, but is a relatively insensitive technique for detecting advanced cervical disease.78 A malignant finding on peritoneal washing in patients harboring ovarian carcinoma is of major importance for substaging this type of neoplasm, especially early (FIGO I and II) ovarian cancer, where presence of malignant cells warrants assignment to stage IC or IIC and indicates a poorer prognosis (Fig. 25).76 Cytology is more sensitive in detecting ovarian carcinoma in ascites than in peritoneal washings, as well as in patients with peritoneal metastasis greater than 0.5 cm.

  Fig. 25. A and B: Serous papillary tumor cells found in pelvic washing (A, Papanicolaou, and B, Diff-Quik, ×400). C: Cell block (H & E, ×400). D: Borderline serous papillary tumor of ovary. (H & E, ×400)



The presence of epithelial cells in the peritoneal fluid of patients with borderline/atypical proliferative ovarian tumors has previously been a problematic area in cytopathology. This problem no longer exists because accurate subclassification of neoplasms into benign and malignant types renders the peritoneal cytology specimens in most of the patients who have benign tumors to be of no relevance. The small number of patients with bona fide carcinoma (patients with micropapillary serous carcinoma or atypical proliferative serous tumor) can be substaged based on the presence or absence of epithelial cells resembling the primary ovarian tumor in the cytology specimens.

Between 5% and 15% of patients with endometrial carcinoma have positive peritoneal cytology, and this may be the only manifestation of extrauterine spread of the tumor. The presence of malignant cells in the peritoneal sample, qualifies for stage IIIA. The literature regarding the significance of this spread has varied, with some studies reporting higher recurrence, and others noting lack of significance in predicting recurrence or death.77, 79 However, the weight of evidence in larger studies with multivariate analysis, supports the presence of malignant cells in peritoneal washings of patients with endometrial carcinoma to be a significant predictor of poor prognosis in all disease stages.80

Mesothelial cells collected by saline washings during laparoscopy, laparotomy, or culdocentesis are morphologically different from those desquamated spontaneously. The latter float in peritoneal, pleural, or pericardial effusions and acquire a globular appearance. In contrast, mesothelial cells taken by force in washings are arranged in monolayers assuming a mosaic-like appearance. The nuclei are monomorphic and easily identified as benign cells (Fig. 26). However, in cell blocks of these washings, strips of mesothelial cells assume a coiled appearance during the centrifugation, mimicking papillary-like fronds and acinar-like formations suggestive of adenocarcinoma. Close scrutiny shows the cuboidal, monomorphic appearance of mesothelial cells.

 Fig. 26. A monolayer of benign mesothelial cells from a peritoneal washing. (A: Papanicolaou; B: Diff-Quik, ×600).

It is important to avoid false-positive or false-negative readings for adequate patient management. A false-positive rate of 4.5% has been reported in benign conditions.81

The most important pitfall in the examination of peritoneal cytology specimens in women involves benign epithelial proliferations.67 To avoid erroneous readings, the cytopathologist should be familiar with the morphologic features of benign-reactive, low-grade tumors, and müllerian inclusions and intraperitoneal chemotherapy effect.76, 81, 82 Sneige and associates81, 82 studied eight cases of müllerian inclusion, four of which were misdiagnosed as adenocarcinoma. Several concepts have been offered in the literature to explain the histogenesis of müllerian inclusions, including lymphatically transported endometrial tissue, müllerian metaplasia of the coelomic epithelium,83 and tubular epithelium sloughing secondary to inflammatory processes of the fallopian tube mucosa. Metaplasia of the müllerian epithelium seems to be the best accepted concept. These inclusions have been referred to as benign glandular inclusions, endometriosis, and salpingiosis.81, 82 Histologically, these lesions are tubular or papillary fronds arranged in single layers and embedded in the uterine serosa, omentum, or pelvic lymph nodes.84, 85, 86 The epithelial component resembles the epithelium of the fallopian tubes, endometrium, or endocervix.83 These structures may shed clusters of cells in single rows surrounding psammoma bodies. This feature is a potential source of confusion, and these structures may be diagnosed as well-differentiated adenocarcinoma in peritoneal washings by a less experienced cytopathologist (Fig. 27).81, 82 Another source of confusion is overstaining of cells that are detached from focal hyperplasia of the pelvic peritoneal epithelium or ovarian surface. Globules of collagen surrounded by epithelial cells are characteristic of this entity (Fig. 28A and B).87

Fig. 27. Histologic section of müllerian inclusion showing a structure formed by collagen infiltrated by lymphocytes and showing foci of calcification (Hematoxylin and eosin, × 900). Inset. A psammoma body surrounded by a single row of epithelial cells. (Papanicolaou, ×900).

  Fig. 28A and B. Globules of collagen and mesothelial cells from a patient with hyperplasia of the ovarian surface epithelium. (Papanicolaou (A) and Diff-Quik (B), ×600).

Hyperplasia of the mesothelium associated with calcified structures also may cause confusion because it mimics serous neoplasms with psammoma bodies.67 They are commonly found in specimens obtained from patients with pelvic inflammatory disease. There is lack of atypia, which is characteristic of nonneoplastic processes.

FINE-NEEDLE ASPIRATION OF THE OVARY

Fine needle aspiration (FNA) can be used in ovarian cysts and neoplasms. When both preservation of ovarian function in young women and assessment of the nature and spreading pattern of malignant tumors are necessary, FNA of ovarian neoplasms is indicated. It can also be used in workup of primary neoplasms, biopsy of superficial masses in patients with known prior disease, follow-up of irradiated patients, and follow-up of patients undergoing chemotherapy.88 FNA of ovarian tumors can be performed through the vagina, rectal mucosa, or percutaneously through the abdominal wall and guided by CT scan or ultrasound. This information should be conveyed to the cytopathologist, because when done transvaginally, squamous cells can be seen and be mistaken for a component of a dermoid cyst. FNA has an accuracy of approximately 90% and a low complication rare.88 Occasionally, infection was reported when aspiration was done transvaginally and transrectally.88, 89 Frequently,89, 90 the transpelvic route is favored because in larger cystic ovarian tumors, the solid portion is located intrapelvically, whereas the cystic portion is situated in the abdomen.90

Aspiration of nonneoplastic ovarian cysts usually is performed in lesions that are up to 4–5 cm in diameter. The cysts must be unilocular, translucent, and without semisolid or solid components or surface excrescences. Squamous cells and colonic cells from the vagina, rectum, and mesothelial cells are frequent components of the aspirate. Colonic cells retrieved during aspiration should be identified precisely, and not misdiagnosed as cells arising from a mucoproducing ovarian neoplasm. In many countries other than the United States this approach is used to sample large ovarian lesions. However in this country fine-needle aspiration of ovarian lesions is mainly reserved for small cystic lesions, and in women being investigated for infertility. Only if there is evidence of large ovarian lesions with extension into the pelvis or metastatic disease, fine-needle aspiration is used as a diagnostic tool.

Nonneoplastic Lesions

Nonneoplastic lesions comprise endometrioid, follicular, and corpus luteum cysts.

Surface epithelial inclusions, paraovarian, and paratubal serous cysts yield clear fluid and degenerated epithelial cells. In aspirates, they are indistinguishable from each other, and clinical correlation is necessary to establish their precise nature.91

ENDOMETRIOID CYSTS AND ENDOMETRIOSIS.

Aspirates of endometriosis contain hemosiderin-laden histiocytes and clusters of endometrial cells in varying stages of degeneration, seen against a background of debris. Not all of these elements are consistently present in aspirates from patients with endometriosis. However, the presence of histiocytes and clinical suspicion by the gynecologist point toward this diagnosis. After multiple bleeding episodes the epithelium may become hyperplastic and highly atypical, mimicking adenocarcinoma in cytology samples and possibly result in a false positive diagnosis. To compound the problem some types of uterine (papillary serous) and ovarian (clear-cell) adenocarcinomas can arise in association with endometriosis. Also the presence of extensive adhesions may also mimic ovarian neoplasia radiologically.

FOLLICULAR CYSTS and CORPUS LUTEUM CYSTS.

Most frequently, patients seek medical care because of menstrual disturbances related to increased estrogenic stimulation produced by these cysts.90 Aspiration of follicular cysts yields clusters and single cells with a high nuclear-cytoplasmic ratio and granular chromatin mimicking cells originating from neuroendocrine tumors (Fig. 29). Mitotic figures may be present sparsely or in high numbers.92 Nuclear grooving also may be observed. The differential diagnosis with cystic granulosa cell tumors is based on the identification of Call-Exner bodies. They are present in cystic granulosa cell tumors and absent in cells derived from follicular cysts. Luteinized follicular cysts may show nuclear atypia in postpartum patients and also in young, cyclic patients.92

Aspiration of corpus luteum cysts may show luteinized cells (granulosa), which have abundant foamy or granular cytoplasm, and small, eccentric and oval or round nuclei (Fig. 30A and B). Atypica can be seen.

Fig. 29. Histologic section of a portion of a follicular cyst showing layers of follicular cells lining the luminal surface. (Hematoxylin and eosin, ×680). Inset. A cluster of follicular cells in three-dimensional arrangement showing limited cytoplasm and enlarged nucleoli. (Papanicolaou, ×900).

  Fig. 30. Corpus luteum cyst. A: FNA (Diff-Quik, x600). B: FNA (Papanicolaou, ×600). C: Histologic section (Hematoxylin and eosin, ×100). D: Histologic section (Hematoxylin and eosin, ×600).



 

  

Neoplastic Cysts

Fine-needle aspiration of neoplastic cysts is limited to tumors with adhesions to the pelvis, caused by malignant infiltration and tumors with known metastasis. Mobile cystic lesions larger than 4 cm usually are not aspirated because they may represent neoplastic processes that when punctured, may seed the pelvic peritoneum.

Benign neoplasms and tumors of low malignant potential are difficult to evaluate on cytology because of sample restriction and lack of the architectural criteria encountered in histologic sections. Benign tumors usually yield monolayers of monomorphic benign-appearing cells.

Usually, borderline tumors are more cellular, with variably shaped nuclei (Fig. 31) and more pronounced nuclear atypia. It is difficult to differentiate borderline tumors from invasive carcinomas by cytology alone in the majority of cases, and a cytologic diagnosis of adenocarcinoma maybe be rendered in a tumor that is borderline on histology.

  Fig. 31. Borderline serous papillary tumor of ovary. A: Cystic fluid cytospin (Diff-Quik, ×600). B: Cystic fluid cytospin (Papanicolaou, ×600). C: Histologic section (Hematoxylin and eosin, ×400).

Serous, mucinous, endometrioid, and clear cell tumors of the ovary are for the most part cystic lesions, and aspirates usually yield some fluid. The most common neoplasm is the serous type, which yields groups of cells in papillary, acinar, and single forms. Psammoma bodies may be encountered in aspirates, but this feature is present in only a minority of cases (Fig. 31).93 Mucinous tumors yield a variable amount of mucin and maybe paucicellular. Mucinous cells can also resemble histiocytes. Well-differentiated mucinous malignant tumors are difficult to differentiate in aspirates from borderline and benign mucinous neoplasms of the ovary (Fig. 32). Clinical correlation and location of the lesion may shed some light in the differential diagnosis.

  Fig. 32. Mucinous cysadenoma of ovary. A. Cystic fluid cytospin (Diff-Quik, ×600). B: Cystic fluid cytospin (Papanicolaou, ×600). C: Cystic fluid cell block (Hematoxylin and eosin, ×600). D: Histologic section (Hematoxylin and eosin, ×600).

Aspirates from endometrioid adenocarcinoma of the ovary can mimic serous type neoplasms in aspirates. Because psammoma bodies are not present with great frequency in aspirates of the serous type (Fig. 31), and endometrioid carcinomas also may have psammoma bodies their presence is not a marker for differentiating these two neoplasms.93 Sex-cord stromal and germ cell tumor components can be identified and diagnosed on cytology, however accurate classification is dependant on the adequacy of sampling.

Metastasis to the ovary most frequently originate from the gastrointestinal tract and the breast. Identification of the tumor as adenocarcinoma is easily done on cytology, however establishing the origin may require immunohistochemical stains and clinical correlation. Signet ring cell type of morphology is often seen in Krukenberg tumors. In cases in which a primary tumor has been diagnosed previously, comparison of the histologic features with those of the primary tumor can establish whether the cells aspirated from the ovary are part of a primary or metastatic process.89 Lymphoma in the ovary is, for the most part, a secondary/metastatic neoplasm.

Fine-Needle Aspiration Cytology in the Staging of Gynecologic Malignancies

Under radiologic guidance, fine-needle biopsy biopsy is used in gynecologic oncology in the evaluation of intra-abdominal and pelvic masses and also for any superficially palpable masses/lymph nodes. Fine-needle aspiration can be utilized for primary diagnosis or staging/recurrence/detection of metastasis in various malignancies. Fine-needle aspiration biopsy for detection of metastatic disease has a high sensitivity and specificity.94 The cytologic appearance of metastatic gynecologic malignancies is similar to the morphology of the primary tumor.

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