Cytohistology, colposcopy and in situ hybridization in cervical preneoplastic lesions: A correlative study

Inr J Gynrcol

Ohsrer.

International

Federation

323

1991, 35: 323-326 of Gynecology

and Obstetrics

Cytohistology, colposcopy and J situ hybridization preneoplastic lesions: a correlative study N. Pasetto,

(Received

January

E. Piccione,

31%

L. Mantenuto

in cervical

and F. Sesti

1990)

(Revised and accepted August

15th. 1990)

Abstract Formalin-fixed, paraffin-embedded cervical tissues from 106 patients which exhibited u colposcopically atypical transformation zone, were examined for the presence of HP V DNA by in situ hybridization using biotin-labeled HP V 6/l I and 16/18 DNA probes. From the comparutive study between histologic, cytologic, colposcopic findings and virologic results it is tionfirmed that HPV 6/l I DNA is detected more frequently in the less severe lesions, whereas HPV 16/18 DNA is predominantly found in the most severe dysplastic lesions.

specimens, with the limits due to the fact that koilocytosis and diskeratocytosis represent a late cytopathic viral effect, with subsequent high incidence of false negative results and the impossibility of defining the implied viral type. Due to recent progress in molecular biology, hybridization techniques are now available not only to identify specific viral genomic sequences, but also to differentiate between the various types of HPV. Thus, the aim of the present clinical trial was to define the possible correlations between cytohistologic and colposcopic changes and specific genomic viral sequences, identified by the in situ hybridization technique using biotinlabeled probes. Materials and methods

Keywords: Human papillomavirus; hybridization; Cervical preneoplastic

In situ lesions.

Introduction Clinical studies [ 1,7,8] about the possible oncogenetic role of human papillomavirus (HPV) in the development of cervical neoplastic lesions, have pointed out the necessity to improve the virologic diagnosis in order to identify the most oncogenic viral types. So far, the traditional diagnosis of cervical viral infections was essentially found on the histopathological reading of cytohistologic

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1991 International

Published and Printed

At the Outpatient Clinic of Cervical-Vaginal and Vulvar Diseases, Department of Obstetrics and Gynecology, IInd University of Rome, the present clinical study of HPV DNA identification and typing was carried out on cervical biopsy specimens, derived from 106 patients who exhibited a colposcopically atypical transformation zone. These patients were selected among 1456 women examined from January to December 1989. The technique for in situ hybridization with biotinylated probes of the paraffin-embedded tissues is described in detail elsewhere [5,11].

Federation in Ireland

of Gynecology

and Obstetrics

trnd Clinictrl

Rcsc~rd~

324

Pusurro

et ul.

from 0 to 8 points was attributed, and their sum has provided a total of: (a) O-2 points, indicative of the less severe lesions (borderline changes and SPI/CIN 1); (b) 3-5 points, indicative of intermediate degree lesions (CIN l-2); (c) and 6-8 points, indicative for aneuploid lesions (CIN 2-3). The cytohistological alterations were classified as follows: (a) in the first group, equivocal changes, such as iperparakeratosis, basal hyperplasia, papillomatosis, acanthosis (possible precursors of the flat condylomas) [lo] were included; (b) in the second group, the so-called flat condylomas or subclinical papillomaviral infection (SPI) and CIN 1 lesions were included; (c) in the third group, CIN 2-3 lesions were considered. The correlations between the observed results were statistically analyzed by chi-square test.

Briefly, sections of 4-6 pm from formalinfixed, paraffin-embedded tissues were placed on adhesive ATP-treated slides and incubated at 60°C for 1 h. Tissue sections were deparaffined and treated sequentially with digitonin (0.05X, 23°C 5 min), water, proteinase K (5 &ml, alcohol dehydration and RNase A (100 &ml) and RNase T (10 units/ml) at 37°C for 30 min. Hybridization solution containing biotinlabeled HPV 6/l 1 and 16/18 DNA probes was pipetted onto the sections, which were then covered with siliconized coverslips. The slides were baked at 92°C for 10 min in a heating block and then incubated at 37°C for 30 min. Streptavidin alkaline phosphatase conjugate was pipetted onto the sections, which were then incubated at 37°C for 15 min in the heating block. This was fol!&ed by the addition of 3-amino-9-ethylcarbazole (AEC) and incubation at 37°C for 15 min. The slides were washed in phosphate-buffered saline, air dried and examined at 40-100x under a light microscope. Criteria for positive results included a red/purple stain located primarily in the nuclei. Positive control probes were used on each tissue biopsy to ensure that tissue sections were properly fixed, digested and stained. Reproducibility was confirmed with duplicate adjacent tissue section. Every patient was subjected to a cytologic (Pap-test) and colposcopic examination, with evaluation of the four signs of Reid’s colposcopic index [9]. To every patient, a score

Results

Table 1 shows the correlation between cytologic, colposcopic, and histologic findings. The correspondence between cytology and histology has been 76.42% (81 out of 106 patients), whereas the colposcopic score was well correlated to histology in 90.57% of the cases (96 out of 106 patients). HPV DNA was detected in 12 cases (23.10%) of equivocal histologic changes, 27 cases (81.80%) of SPI/CIN 1 and 15 cases (71.40%) of CIN 2-3. The presence of HPV 6/l 1 DNA was significantly correlated to the less severe borderline and SPI/CIN 1 lesions, whereas HPV

Table 1. Correlation between cytologic, colposcopic, and histologic findings. Histology

Total

Colposcopic score

Cytology Negative

SPII CIN I

CIN 2-3

t&2

3-5

49 I9 -

3 I3 2

I I9

51 31 6

I I 2

-

SPIKIN 1 CIN 2-3

I I3

52 33 21

Total

68

I8

20

88

4

I4

106

W

Inr J Gymcot Ohstel 35

HP V DNA

Table 2.

Correlation

findings.

x’ = 69.587.

Histology

between

Egv

IO I

CIN 2-3 Total

results

and virologic

HPV DNA negative

HPV DNA positive 611 I

SPIKIN

cytologic

P < 0.001.

Total

dings.

Correlation

2 3

40 6

52 33

2

13

6

21

36

I8

52

106

between cytologic

results and virologic tin-

x’ = 50.20. P < 0.001.

Cytology

HPV DNA negative

HPV DNA positive 611 I

Colposcopic score (points)

HPV DNA positive 6/l I

24

Total

16118

Negative or equivocal

23

2

43

68

SPKIN I CIN 2-3

II 2

3 I3

4 5

I8 20

Total

36

18

52

106

preneoplustic’ lesions

Table 4. Correlation between colposcopic findings. x’ = 39.268, P < 0.001.

16118

16/18 DNA was significantly associated with the most severe CIN 2-3 lesions (~2 = 23.232, P < 0.001) (Table 2). From the correlative study between cytologic and virologic findings, it has resulted that HPV DNA was detected in 25 cases (36.76%) of negative or equivocal cytologic results, 14 cases (77.77%) of SPIKIN 1 and 15 cases (75.0%) of CIN 2-3. Also the cytologic lindings have confirmed the correlation between HPV 6/l 1 DNA and negative or equivocal changes and definite minor grade lesions (SPUCIN 1) and between HPV 16/18 DNA and suspected high grade lesions (CIN 2-3) (x2 = 9.964, P c 0.005) (Table 3). The comparative analysis of the colposcopic and virologic findings has shown that HPV DNA was detected in 40 cases (45.45%) with colposcopic score of O-2 points, 3 cases (75.0%) with intermediate score (3-5 points) and 11 cases (78.57%) with the highest score Table 3.

types in wrsicul

325

score and virologic

HPV DNA negative

Total

l6/18

o-2 3-5 6-n

34 I I

6 2

48

I

88 4

IO

3

I4

Total

36

I8

52

106

(G8 points). The presence of HPV 6/l 1 DNA was significantly associated with lesions with the lowest colposcopic score, whereas HPV 16/18 DNA was predominantly correlated to lesions with the highest score (xl = 19.694, P < 0.001) (Table 4). Discussion

The major objective of this investigation was to determine whether equivocal cytohistologic changes and definite cervical preinvasive lesions of various grade of severity are associated with any specific HPV types, identified by in situ hybridization with biotin-labeled probes. The results indicate that HPV DNA is frequently (23.10%) associated with equivocal histologic changes, such as basal hyperplasia, papillomatosis, acanthosis, iperparakeratosis. This fact confirms the presence of HPV DNA at an early stage, in which the typical alterations of the flat condylomas (koilocytosis and dyskeratocytosis) are not yet produced [ 1, lo]. The typical alterations of HPV infection (SPI) and CIN 1 lesions appear to be more strikingly associated with HPV 6/l 1 DNA. The HPV 16118 DNA was distributed in a pattern exactly opposite to that of HPV 6/l 1, because it was strongly associated with the most severe CIN 2-3 lesions. Thus, these data indicate that the patterns of association of HPV 6/l 1 and HPV 16/18 DNA in cervical lesions are different and HPV 16118 infections are more likely to progress to neoplasia [2,3,4,6,12]. Clinical

und Clinical

Reserrrch

The presence of HPV DNA in a significant rate (36.76%) of negative or equivocal cytologic changes confirms the low sensitivity of cytology in the early diagnosis of HPV infections [lo]. This is also shown by our elevated rate (57.58%) of false negative cytologic results (19 out of 33 patients with histologic diagnosis of SPI/CIN I) (Table 1). The distribution of HPV types in cytologic alterations was very similar to that in histologic changes (Table 3). Comparative analysis between the score from the colposcopic index and the distribution of HPV types has provided a pattern of association quite analogous to that of the histologic-virologic correlations (Table 4). Considering also the good correlation between the colposcopic score and histology (Table l), the value of this colposcopic grading as a less subjective approach to the colposcopic differentiation of minor-grade atypia from areas of high-grade colposcopic change is confirmed. Thus, the in situ hybridization test of routinely collected and pathologically wellcharacterized material can provide valuable information. However, the test has several limitations. Preliminary studies with biotinylated DNA probes suggest that from lO@-800 copies of target DNA/cell are required for a positive signal [7]. Thus, biotin-labeling has the advantage of avoiding radioactivity, but the sensitivity of detection is 10 times lower than that of isotope-labeled probes. This relative lack of sensitivity probably results in underdiagnosis of HPVs which replicate less efficiently and produce low copy numbers. Second, we employed only two HPV probes, although several additional HPVs (HPV-31,33,35, 39 and 42-44) are known to infect the genital tract. A proportion of the hybridization negative group can represent infection with HPVs other than the ones used as probes. In conclusion, the use of in situ hybridization with biotinylated probes appears advantageous to identify and localise HPV infected cells whilst simultaneously evaluating the morphology of infected and noninfected cells, and to make retrospective study of HPV DNA in paraffin-embedded tissues. However, InrJ

Gynecol Ohster 35

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Address for reprints: N. Pasetto Department of Obstetrics and Gynecology Ilnd University of Rome Hospital S. Eugenio Piazzale Umanesimo 00144 Rome, Italy