Cytological variations and typical diagnostic features of endocervical adenocarcinoma in situ: A retrospective study of 74 cases

Subjects

We retrospectively recruited 74 cases of cervical conization or hysterectomy materials with stage 0 cervical AIS that were diagnosed during a 20-year period from 1993 to 2012 at the Department of Pathology, Jikei University Hospital, Tokyo, Japan. The mean age (range) of the 74 subjects was 41.2 years (21–69 years). The surgical treatments were as follows: Cervical conization alone in 47 cases, simple hysterectomy in 22 cases, and conization and subsequent hysterectomy in five cases. Cases with microinvasive or invasive adenocarcinoma were excluded from the present study. Endocervical smears were usually taken using a cytobrush by gynecologists before surgery. A total of 140 Papanicolaou smears were reviewed. All the smears were prepared using the conventional method: The smears were fixed in 95% ethyl alcohol and were stained with the standard Papanicolaou stain. No liquid-based preparations were used.

Cytological review

All the cervical papanicolaou smears were first blindly rescreened individually by six cytotechnologists including the authors without knowing the final histological diagnosis. If there was discordance among the cytological diagnosis, the consensus meeting was held to reach the final diagnosis. Initial and review papanicolaou smears were diagnosed according to the TBS2001 system.[34] We analyzed the diagnostic accuracy of AIS and the factors influencing the sampling/screening/diagnostic errors. Sampling errors were defined as cases with smears that were initially and finally reported as negative for intraepithelial lesion or malignancy (NILM). Screening errors were defined as cases with possible or definite high-grade epithelial abnormalities initially reported as NILM. Diagnostic errors were defined as AIS cases initially reported as atypical glandular cells (AGC; underdiagnosis), low-grade squamous intraepithelial lesions (LSIL/HSIL; underdiagnosis), adenocarcinoma (overdiagnosis) or SCC (overdiagnosis).

The tissue fragments of abnormal cells were called microbiopsy or hyperchromatic crowded groups (HCG).[1516] HCG are three-dimensional clusters of crowded cells with hyperchromatic nuclei and a high nuclear/cytoplasmic ratio. We analyzed the presence or absence of detailed cytological features, such as feathering, mitosis, polymorphism, endocervical samples, and necrotic diathesis. We designated well-differentiated AIS as typical AIS and poorly differentiated AIS as polymorphic AIS.[5] When features of both typical and polymorphic were observed, the term mixed typical and polymorphic AIS was applied. Thus, three distinct differentiation patterns of AIS were applied to our series of samples: Typical AIS, mixed typical and polymorphic AIS, and polymorphic AIS. We also classified the AIS into three subtypes: Endocervical, intestinal and endometrioid subtypes. No Paneth cell subtypes were included among the samples.

Sampling/screening/diagnostic errors of the cytological diagnosis

Normal endocervical samples were included in all the Papanicolaou smears; thus, the specimens were suitable for the cytological diagnosis of the endocervix. The overall flow chart of the cytological diagnosis before and after the review is shown in Figure 1. Glandular abnormality was absent in two cases (one each case of HSIL and NILM). The review showed AGC in one case of NILM. Thus, sampling and screening errors were only present in one case each (1.4%). The final cytological diagnosis of glandular abnormality included AIS in 58 cases (78.4%), AGC in 11 cases (14.1%), and adenocarcinoma in 3 cases (4.1%). A total of 25 cases were initially misdiagnosed as other cytological diagnoses than AIS. These misdiagnosed cases are shown in red boxes in Figure 1. The diagnostic error rate was 33.8% and the total error rate was 36.5%.

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Figure 1

Flow chart of 74 pathologically-confirmed adenocarcinoma in situ cases before and after a cytological review. The left column shows the distribution of the initial cytological diagnoses, and the right column shows that of the final cytological diagnoses after the review. A few sampling and screening errors occurred, but diagnostic errors predominated. AGC: Atypical glandular cells, AIS: Adenocarcinoma in situ, HSIL: High-grade squamous intraepithelial lesion, LSIL: Low-grade squamous intraepithelial lesion, NILM: Negative for intraepithelial lesion or malignancy, SCC: Squamous cell carcinoma

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Cytological details of glandular abnormalities

The cytological features of 72 cases with glandular abnormality are shown in Table 1. The common characteristic features were microbiopsies/HCG (82.0%) and mitotic figures (72.2%). The appearance of single cells (2.8%) was rare, and all the cervical cytology smears lacked necrotic tumor diathesis.

Table 1 Retrospective cytological features of 72 cases* with endocervical glandular abnormalities

Among 58 cases with AIS, 41 cases showed a typical AIS pattern (67.2%). The remaining 17 cases (32.8%) of AIS showed the apparent polymorphism in some cancer cells, then diagnosed as mixed typical and polymorphic AIS. Typical histological and cytological figures of polymorphic AIS are shown in Figures 2 and 3. Three cases of purely polymorphic AIS were cytologically diagnosed as (invasive) adenocarcinoma. The distribution of the AIS subtypes is shown in Table 2 and typical cytological figures of AIS subtypes are shown in Figure 4. The endocervical subtype was the most predominant subtype, followed by a few mixed subtypes. The pure endometrioid subtype was present only in one case.

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Figure 2

Histological picture of endocervical polymorphic adenocarcinoma in situ. Note large polymorphic nuclei with conspicuous nucleoli. No stromal invasion is present (H and E stain, ×400)

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Figure 3

Cytological pictures of endocervical polymorphic adenocarcinoma in situ (AIS). (a) The tissue fragments of typical endocervical AIS. Dense clusters of darkly stained AIS cells in microbiopsies/hyperchromatic crowded groups (HCG) are present. The AIS cells at the margin of the clusters show no feathering (Papanicolaou stain, ×400). (b) Polymorphic AIS. The AIS cells have large polymorphic nuclei with prominent nucleoli (Papanicolaou stain, ×400). (c) Polymorphic AIS. The AIS cells often exhibit mitotic activity (arrows) (Papanicolaou stain, ×600). (d) Single or loosely arranged AIS cells in polymorphic AIS. Microbiopsies/HCG or three-dimensional clusters are scarce in this case (Papanicolaou stain, ×600)

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Table 2 Subtypes and differentiation patterns of 61 cases with endocervical AIS (n=58) or adenocarcinoma (n=3)

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Figure 4

Typical cytological features of adenocarcinoma in situ. (a) Endocervical subtype. Pseudostratified strip of cells demonstrating crowding, nuclear enlargement and peripheral feathering (Papanicolaou stain, ×600). (b) Endometrioid subtype. Pseudostratified cluster with nuclear crowding, irregular internuclear distances, and scattered mitotic figures (yellow arrow heads) (Papanicolaou stain, ×600). (c) Intestinal subtype. Note large mucin droplets in the clusters (Goblet cells) (Papanicolaou stain, ×400)

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Among 11 cases with a final cytological diagnosis of AGC, cytological features favoring a diagnosis of AIS were infrequent; microbiopsies/HCG and frequent mitosis (3 cases each, 27.3%). Abnormal clusters were often obscured by large amounts of blood (one case) or mucus (three cases) and severe inflammation (two cases). Numerous HSIL cells coexisted with AGCs in two AGC favor neoplastic (FN) + HSIL cases, masking glandular abnormalities.

Cytological features of adenocarcinoma in situ with diagnostic errors

The cytological features of 25 AIS cases with diagnostic errors are shown in Table 3. The polymorphism was frequently marked among nine cases with an initial diagnosis of (invasive) adenocarcinoma (5/9 cases). In eight cases with the initial diagnosis of SIL (one case of LSIL and seven cases of HSIL), HSIL frequently coexisted (6/8 cases).

Table 3 Cytological features of 25 misdiagnosed cases* with final cytological diagnosis of AIS

Characteristic cytological pictures of adenocarcinoma in situ

The present study showed that the characteristic cytological features of AIS included microbiopsies/HCG, mitotic figures of the cancer cells, and a lack of necrotic tumor diathesis in polymorphic AIS.

Causes of misdiagnosis

When abnormal glandular cells in a sample are scarce, the diagnosis of AIS is often difficult and may result in a diagnosis of NILM, rather than AGC.[51011] These cases should be diagnosed as AGC-FN to prevent AIS from being overlooked.[4] The Update on ASCCP Consensus Guidelines recommends an excisional procedure for AIS if the initial cytology is AGC-FN or AIS and no invasion is identified.[20] Only seven cases in this study exhibited AGC-FN. Faraker and Boxer indicated that the false-negative factors in cervical cancer screening were (1) scant abnormal cells and (2) abundant dyskaryotic cells presenting as microbiopsies/HCG.[21]

Limitation of the study

The retrospective nature of this study limits the implication of the results. As we have introduced the liquid-based cytology (LBC) system in our lab shortly after this study, we could not reproduce our results in a prospective way. This study adopted the conventional Papanicolaou method rather than LBC. The LBC system has already been widely introduced in US and other western countries, but the conventional method is still widely used in developing countries, because they could not introduce the LBC system due to the financial limitation. The introduction of LBC system has been delayed in Japan as well (<10%). As the cytological details are slightly different between the conventional method and LBC, we safely conclude our results are at least applicable to the cytology using the conventional Papanicolaou method.