CYTOLOGY-EDUCATIONAL NOTES

INVASIVE ENDOCERVICAL ADENOCARCINOMA

Columnar cells possess cytoplasmic vacuoles, nuclear enlargement, eccentric nuclei, large eosinophilic nucleoli, multiple nucleoli and papillary spherical clusters. There is overlapping of nuclei and clusters may have a lumen (rosette or glands). Such cells are seen in adenocarcinoma, squamous cell carcinoma and HGSIL.

It is difficult to differentiate between dysplastic and malignant endocervical cells. However, in adenocar- cinoma, smear shows necrotic and bloody background.

Signet ring cells may be present. Mitotic figures and apoptotic nuclei are present. A few dysplastic squamous cells may be seen (Fig. 16.38).

OTHER

This category is created for reporting normal or abnormal endometrial cells in women who are 40 years or older, as the presence of even benign-appearing endometrial cells on cervical cytology in women who are at least 45 years of age is more often associated with endometrial adenocarcinoma and endometrial hyper- plasia than with benign endometrium. However, since cervical cytology is primarily a screening test for squamous epithelial lesions and squamous cancer, it is not reliable for detection of endometrial lesions.

ANCILLARY TESTING AND EDUCATIONAL NOTES

If slides are scanned by automated computer systems, the type of system used should be reported with the result. Ancillary testing such as HPV DNA is performed, if appropriate, and reported with the cytology results.

Written comments regarding the interpretation of a cytologic specimen are optional and may be conveyed to the clinician by the pathologist as a means of clarification and information.

CYTOLOGY-SPECIMEN ADEQUACY

SATISFACTORY FOR EVALUATION

Cervicovaginal smear should be representative of epithelia of ectocervix, endocervix and should include transformation zone as aim of cervical smear examination is detection of abnormal cells which generally arise in the transformation zone. Therefore, ideally the smear should contain squamous cells, metaplastic cells (TZ) and/ or endocervical cells, and should not be obscured by the presence of blood/ inflammation, etc. However, since the absence of endocervical cells/presence of partially obscuring factors have not shown to increase the risk of a false negative report; TBS 2001 considers such specimens to be reported as “satisfactory for evaluation”, but the comments about the TZ components/ partially obscuring factors are placed in the narrative report which should be read carefully by the clinician to note that the trans- formation zone was not sampled and to improve the specimen adequacy henceforth. In conventional Pap smears, cervical mucus itself is evidence of smear adequacy in absence of endocervical cells. However, in liquid-based preparations, this valuable resource is lost. Since only a part of sample aliquot is used in smear preparation, 5000 well preserved squamous cells per smear and 10 endocervical cells form the basis of adequacy. In older women, TZ may be situated within endocervix canal and is much more difficult to sample and presence of endocervical cells or mucus is not necessary for adequacy. Any smear containing abnormal cells is considered satisfactory for evaluation irrespective of the number of cells present.

UNSATISFACTORY FOR EVALUATION

When the smear does not contain adequate cellular material for reliable interpretation, it is reported as “unsatisfactory for evaluation”. The report would specify, whether the specimen was rejected and not processed (with reason); or whether it was processed and examined, but found unsatisfactory for evaluation of epithelial abnormalities. The causes of unsatisfactory smears could be:

  1. Heavy inflammation obscuring cells (Fig. 16.14)
  2. Air drying artifact (Fig. 16.15)
  3. Excessive cytolysis (Fig. 16.16)
  4. Bloody smear with only few cells
  5. Uniformly thick smear
  6. Scanty cellular material
  7. Lubricant contaminant
  8. Inadequate fixation
  9. Unrepresentative material
  10. Menstrual cells

HISTOPATHOLOGY OF PRECANCEROUS LESIONS

HISTOPATHOLOGY OF PRECANCEROUS LESIONS

Histological features that are used to establish diagnosis of precancerous lesions are cellular differentiation and maturation, organization, polarity, nuclear abnormalities and mitotic activity.7,8,26
In normal epithelium, squamous cells are arranged in well defined layers of basal, parabasal, intermediate \ and superficial cells with increasing cytoplasm and a more organised arrangement towards the surface as cells mature. These features are disturbed in CIN and cells lack differentiation with retention of features of parabasal cells, lack organization and polarity as cells in upper layers do not remain parallel to the surface, rather arrange vertically or haphazardly.
The proportion of epithelium showing disturbance of maturation and differentiation reflects the severity of abnormality.
Nuclear abnormalities are characterized by increased nuclear cytoplasmic ratio, hyperchromasia, irregularity in size and shape (pleomorphism and anisonucleosis), coarse chromatin clumping and multinucleation. Severity of these changes will determine the grade of the lesion (Figs. 9.1 and 9.2).
Generally there is a good correlation between the proportion of epithelium showing disturbance of maturation and degree of nuclear abnormality. However, when there is lack of correlation, degree of nuclear abnormality is more reliable for grading.
Abnormality in mitotic activity is assessed by the number, location and type of mitotic figures.Normally mitotic figures are infrequent and are seen only in parabasal layer. In precancerous lesions, the number of mitotic figures increases and these may be found higher up in the epithelium. Mitotic activity is an expression of immaturity of the cells. HPV’ infection increases the number of mitotic figures and may result in attributing an erroneously high grade to the lesion. In CIN, mitotic figures may have abnormal configuration and are important criteria in grading of the lesion. The most common aberration is the three group metaphase (triaster). Mitotic figures with more than two spindles (quadraster or multipolar mitotic figures) and/ring mitotic figures are also frequently seen.
Keratinization can sometimes be seen on the surface of CIN generally of higher grade (Fig. 9.3).

LOW GRADE CIN/SIL (CIN I)

Abnormal cellular changes are mild and are confined to the lowest one third of the epitheliurn.Y? There is usually good maturation of upper layers. Nuclear abnormalities are mild and are confined to the deeper layers of epithelium. However, some abnormalities persist at the surface also; otherwise cytological recognition of low grade lesions will not be possible. Mitotic figures are increased in number, confined to deeper layers and abnormal configurations are not present. HPV cytopathic effects characterized by perinuclear cytoplasmic vacuolation, nuclear hyperchromasia and wrinkling, binucleate cells, increased number of mitotic figures may be present in the upper layers of epithelium (Fig. 9.4).
Involvement of upper layers by HPV changes should not lead to diagnosis of higher grade of lesion.

HIGH GRADE CIN/SIL (CIN 11, ILL)

Maturation and stratification may be present in the upper .1-2 layers of the epithelium or may be completely lacking. Nuclear abnormalities are more marked and extend to the upper layers even up to surface. Marked anisonucleosis and bizarre nuclear forms may be seen. Mitotic figures are markedly increased in number and abnormal forms are frequent. It is to be borne in mind that all epithelia will not show the changes to the same degree. While some epithelia may show marked nuclear pleomorphism and hyperchromasia but low mitotic activity, other cases may show numerous mitotic figures with abnormal forms but little nuclear abnormality (Fig. 9.5). Sometimes intermediate grades of lesions or lesions with mixed picture may be difficult to classify (Fig. 9.6).

LOCATION AND SPREAD (CRYPT INVOLVEMENT)

Preneoplastic lesions develop in the transformation zone, the field of columnar epithelium that undergoes squamous metaplasia. It follows the contours of original columnar epithelium and affects the surface of ectocervix and cervical canal as well as glands/crypts (Fig. 9.7). Very rarely it may involve original squamous epithelium.
Generally higher grade of CIN is seen in proximal location in cervical canal and lower grade caudally towards vagina thereby indicating the importance of endocervical brush or curettage for detection of higher and more severe lesions. As is obvious, cytology of surface lesion or lower end of cervical canal by Ayre’s spatula may miss the CIN lesion or may reveal low grade of lesion only.
Crypt involvement is quite high and was reported in 88.6% of cone biopsies examined for CIN in a study.” Few crypts or only a part of crypt may be involved and uninvolved crypts can be recognized lying alongside (Figs. 9.8 and 9.9). Maximum depth up to 5.2 mm is involved which is the depth \JP to which normal crypts are present. Area and depth of crypt involvement increases with severity of CIN.I
Crypt involvement can sometimes be confused with early invasive carcinoma (Fig. 9.7). Recognition of basement membrane around the cross-section of crypts, circumferential fibrous thickening of stroma surrounding crypts, absence of loose stroma or lymphocytic reaction in the stroma and presence of uninvolved crypts nearby help in differential diagnosis.

DNA PLOIDY AND HPV DNA IN PRENEOPLASIA

It is possible to objectively classify CIN by nuclear DNA ploidy patterns using static or flow cytometry.Condylomas and majority of CIN I have predominantly diploid and occasionally polyploid pattern. 10,24
Aneuploid pattern is seen in one third of CIN I, more than three fourth cases of CIN Il and almost all cases of CIN Ill.
Diploid and polyploid lesions are more likely to regress or persist in contrast to aneuploid lesions which have more propensity for progression.Morphologically abnormal mitotic figures form useful marker of aneuploidy.P Diploid or polyploid lesions are likely to retain polarity; nuclei are relatively uniform and lack abnormal mitotic activity. CIN,with aneuploid DNA in general reveal high cellularity,cellular disorganization, nuclear pleomorphism and high mitotic activity including abnormal forms (Fig. 9.10). 85% of CINs with aneuploid DNA pattern contain abnormal mitotic figures, while less than 20% of CIN with diploid DNA have abnormal mitoses. Aneuploidy increases with severity of the lesion.24.54
Abnormal mitotic figures are also linked to high risk HPV 16 in 80% of cases.25,54 Kadish et al. (1992) demonstrated that abnormal mitotic figures in 12.5% of CINs were related to HPV type 6/11, 78.3% to type 16, 20% to type 18, 21.4% to type 31 and 100% to type 33.37 Lesions associated with high risk HPV types are likely to progress while lesions with type 6/11 regress or persist.s”
Thus from viewpoint of clinical outcome, study of DNA ploidy and HPV typing is important in CIN lesions from the point of management since histology alone cannot always predict biologic behavior of the lesion and the type of associated HPV.