Electrosurgical loop excision/conisation for cervical intraepithelial neoplasia in an algorithm that excludes punch biopsy - a study of 210 cases

Introduction
Electrosurgical loop/conisation are excisional procedures that allow the simultaneous histopathological diagnostic and treatment of the cervical dysplasias (1).
Thus, they can eliminate a supplementary step represented by punch biopsy and endocervical curettage (ECC). These last two procedures would lead to a certain number of patients being lost of the evidence between punch biopsy, ECC and electrosurgical treatment. It is also possible to avoid diagnostic errors generated by an insufficient specimen offered by the punch biopsy and ECC or by the incapacity of the colpos-copist to identify the most severe aspect for the punch biopsy. In the absence of a national screening programme, since 1980 Romania has a high and increasing rate of incidence and mortality by cervical cancer (2); by using this algorithm, we could prevent a treatment delay.
There are randomized studies in the literature which compare the results of the colposcopy directed punch biopsy followed by LLETZ/electrosurgical conisation with the results of a direct treat by LLETZ/conisation and all of them conferred credibility for the last (3-13).

Objectives
The objective of this study is to evaluate the efficiency of the LLETZ/electrosurgical conisation as a diagnostic and treatment method at the end of a algorithm for CIN including: pap smear, colposcopy and HPV genotyping alone or associated, the histopathological report being taken as gold standard and at the same time excluding from our algorithm the punch biopsy and endocervical curettage (ECC).

Material and methods
Between January 2005 and January 2007, 227 electrosurgical loop/conisation procedures were performed for patients aged between 20-60 years old, with a 36.5 median value.
We excluded 15 cases from these, being lost from the evidence (no control 2 years after conisation) and other 2 patients having an un-interpretable histopathological result.
All patients were subjected to pap smear and colposcopy investigations prior to LLETZ, and only 70 were also tested for HPV typing (as part of a grant study, CEEX 119/2006).
Inclusion criteria:
1. HGSIL smear;
2. LGSIL smear persistent more than 2 years and for at least 3 controls (at 6 months intervals);
3. ASC-H or AGC-NOS smear;
4. ASCUS persistent for at least 3 controls (6 months intervals);
5. normal/benign smear but in discordance with an abnormal colposcopy and/or HPV genotyping of high risk (HR) or probably high risk (PHR).
Exclusion criteria:
1. Pregnancy;
2. ASCUS/LGSIL smears at the first or at the second control;
3. patients with no follow up after conisation;
4. patients with no histopathological diagnosis because of thermal lesions due to electrosurgery.
All patients underwent LLETZ if the transformation zone was type I (TZ I) and conisation with the Fisher loop if the transformation zone was type II, with an unsatisfactory colposcopy or if they had AGC-NOS at the pap smear. Punch biopsy or ECC were not performed before the electrosurgical procedure. For the electrosurgical procedures we used ERBE ICC200 with loops of different sizes and a Fisher loop for the conisation. The viral genotyping was done with INNOLYPA (Innogenetics) kit, with the specific molecular probes for the viral genotypes 6, 11, 16, 18, 31, 33, 40, 45, 51, 53, 54, 59, 60, 68 and 70.
For the investigated patients, we analyzed:
- the over treatment rate (which means normal or benign histopathological result after loop conisation);
- the incomplete treatment rate (which means cases with CIN/invasive carcinoma on the hysterectomy specimen) or CIN persistence at the 6 months-2 years control (pap smear, colposcopy, HR or PHR HPV positive);
- the immediate or late complication rate;
- the artefacts induced by coagulation.
We also analyzed the efficiency of a diagnostic algorithm based on cervical cytology, colposcopy and DNA typing (alone or associated), by comparison with the histopathological loop result, considered as gold standard (over-treatment, omitted lesions, under and over evaluation).

Results
We included in this study 210 patients aged between 20 and 63 with a median age of 36.5, treated by a loop or a conisation procedure for CIN, between January 2005 and January 2007. The Bethesda categories (Fig. 1) included in this study were as follows:
- within normal limits/benign changes 4.29%;
- ASCUS – ASC-H 12.86%;
- LGSIL 55.71%;
- HGSIL 27.14%;
- We didn’t have smears with isolated glandular anoma-lies, but there were 6 cases of AGC-NOS associated with: ASC-H-2cases, ASCUS-2cases, LGSIL-2cases.
Regarding the colposcopic aspects, the investigated subjects were classified according to the Walker international classification (14) (Fig. 2) as:
- in normal limits or benign changes 4.29%;
- with minor abnormalities (grade1-G1) 71.90%;
- with major changes (grade2 -G2) 20.48% - as unique lesion or associated with minor abnormalities;
- unsatisfactory colposcopy, without minor or major abnomalities on the ectocervix 3.33%.
The HPV DNA genotyping revealed that high risk (HR) and probably high risk (PHR) types were present in 70.02% while the low risk types only in 2.85%. HPV negative samples represented 22.85% of the investigated cases. Due to INNOLIPA limits in 3/70 we found inconclusive genotype (HPVx). (Fig. 3)

The repartition of the histological categories (Fig. 4) on the loop histopathological specimen is:
- benign lesion 9.52% as a discordance with cytological results;
- CIN1/flat condilloma 58.09%;
- CIN2-17.62%;
- CIN3/CIS-11.43%;
- microinvasion 2.38% as a discordance with colposcopical findings;
- invasive carcinoma 0.96% as a discordance with colposcopical findings.
Analyzing the efficiency of electrical loop/conisation in this group of patients, we have got the following results:
- the over-treatment rate was 9.52%;
- an incomplete treatment was performed in 20.47%.
From the cases with incomplete treatment, 17 presented CIN or invasive residual disease on the hysterectomy specimen (89.47% from the hysterectomies performed for CIN or invasion), and 26 were patients with an abnormal pap smear of LGSIL/ASCUS at the first control after the conisation (performed at 6 months). But in these 26 cases, 11 became negative at the 2nd control. Only for one case with persistence of ASCUS, positive colposcopy and persistence of HPV 16, 31, after the 3rd control, we performed a 2nd loop and the histopathological result was residual CIN1. (Table 1, Table 2)
In conclusion, we found residual lesion in 14.76%. From these, residual CIN2+ was detected in 3.80% and residual CIN3+ was detected in 2.38%.
We had the following complications: 3 hemorrhages needing hospitalization, the stenosis of the cervical os in 2 cases which made difficult the sampling of the cytology. No patients presented infections after loop/conisation, but the obstetrical prognostic could not be analyzed due to the short period of follow up. Changes due to thermal coagulation were mentioned in 7 reports but only in 2 cases the thermal changes impaired the histopathological interpretation.
The diagnostic efficiency of this algorithm which includes the pap smear, the colposcopy and the HPV genotyping alone or in association, was appreciated in comparison with the histopathological result after the electrosurgical loop or conisation. Thus, we evaluated the rates for the over-treatment, the missed lesions, the over and under evaluation of these lesions. The results are shown in the table bellow: (Table 3)
From the table, we see that the association cytotest-colposcopy is sensible enough, but for a more refined diagnostic and decreasing under and over diagnostic, HPV typing should be added if available.
Nineteen patients underwent hysterectomy. Their distribution according to the pathological result after conisation was: all patients with microinvasive or invasive cancer, 7 cases with CIN3/CIS (with no interest of their reproductive outcome) and one patient with CIN1 and hemorrhagic myoma.
All the patients with microinvasive or invasive results on the conisation specimen were under-evaluated for invasion on the pap smear (HGSIL result) and colposcopy. An explanation could be that the lesions were not very large or they developed into the cervical canal. In the end, all these cases were cervical carcinoma 1A1, 2 or 1B1.
The concordance rate between the histopathological results at the conisation and on the hysterectomy specimen is described in the following table and graphics: (Fig. 5, Table 4).

Discussions
Romania is a country with the highest mortality in Europe and high incidence rate for cervical cancer (2). Without a national screening program and a computerized health system for the population evidence, but with a low level of health education and a poor addressability, a “see and treat “method for the treatment of cervical dysplasias seems to have more efficacy.
In our study, we analyzed the efficiency of the electrosurgical resection/conisation performed for 210 patients who were selected using a diagnostic algorithm which includes the pap smear, the colposcopy and/ or the HPV genotyping, without punch biopsy or ECC (opportunistic triage or patients included into a grant study).
The usual punch biopsy or the ECC could lead to a certain number of under-evaluated or missed lesions, due to the colposcopic inter-observer variability in choosing the most severe site for punch biopsy and also, due to the limited volume of the specimen, sometime insufficient for interpretation. In practice, at least 2 sites are necessary but these increase the volume of the histopathological work and the price of the procedure. (15) Many studies showed therefore that the triage of the patients for LLETZ by an algorithm that includes the pap smear, the colposcopy and the HPV genotyping at the moment of resection, would be better that the punch biopsy (16).This aspect is much more evident for the patients with HGSIL (17-19). The colposcopic directed punch biopsy is not efficient in the treatment of HGSIL lesions because the invasion can not be excluded (12) and, on the other hand, it has a low sensibility for the glandular lesions (13).
The experts’ opinion is that a correct diagnosis of a glandular dysplasia and a correct differential diagnosis with the invasive adenocarcinoma could be performed only on an excisional specimen, including the endocervical canal (conisation).
The European Guidelines for the Treatment of the precancerous cervical lesions (20) recommended in case of colposcopic directed punch biopsy a positive predictive value for CIN1, CIN2 and CIN3 of 16%, 32% and 83% respectively. This is due to the fact that the punch biopsy, even correctly diagnosis the CIN lesions, frequently under-estimate their severity.
HGCIN is under-estimated in 4.3-57% of the cases (8), (10-12).
This fact highlights the subjectivity of the colposcopic exam in the selection of the biopsy sites (21).
The European Treatment Guidelines for the precancerous cervical lesions (12), (20) impose quality standards for the electrosurgical resection/conisation technique: over-treatment (normal histology or benign lesions) <10%; residual lesions <5%; percentage of cases effectuated in outpatient conditions with local anesthesia >80%; >80% to perform a unique specimen for histopathology; the complication rate for primary hemorrhagia (the hemorrhagia which occurs during the procedure and which needs a supplementary hemostasis) <5% and the rate of late complications <2%.
More than 90% of LLETZ must provide an adequate specimen for the histological interpretation.
Analyzing the over-treatment rate, Denny (3) demons-trated that the colposcopic directed punch biopsy does not reduce the percentage of a negative histology after LLETZ. This percentage was 14% for Denny in the arm of the study with the LLETZ treatments without punch biopsy, (n=114) and 18% in the arm with the patients which performed LLETZ after a punch biopsy (n=184).
In our study, the rate of over-treatment (no CIN or invasion on the LLETZ specimen) is 9.5%. Howells (4) finds a percentage of 19% negative histological results after LLETZ in a group of 452 patients. He associates these results with a high frequency of negative cytology or low grade (LGSIL, ASCUS) and with a high frequency of negative or low grade colposcopy similar to our statements, but he found also a high incidence in the 50+ aged group, in contradiction to our data.
Looking for the residual disease, in our study the residual disease rate at the first control was: 4.76% for CIN of any grade, 3.80% for CIN2+ and 2.38% for CIN3+. Kang (22) found a 3% persistence/ recurrence rate after LLETZ for CIN3 (n=101). Kjellberg analyzed 528 conisations for HGSIL performed immediately after a colposcopy without punch biopsy and found a residual rate of 9.4%. (19)
Zlatkov analyzed 114 cases of LLETZ and he found a histopathological repartition of the lesions similar to our study but without invasive lesions. (23) So, the percentage of different histopathological results in the Zlatkov’s study in comparison with our results is the following: benign lesions (7.9% vs 9.52%); CIN1 (58.7% vs 58,09%); CIN2 (20.2% vs 17.62%); CIN3/CIS (11.4% vs 11,43%); micro invasion (1.8% vs 2.38%) and invasion (0 vs 0,96%). In the study, the incidence of the residual disease was 4.9%.
In another study comprising 185 patients treated with LLETZ, Mergui (24) got the following repartition of the histopathological results: CIN1 11.3%; CIN2 23.7%; CIN3 63.7%. The incidence of the residual disease at 1 year was 5.94%. The coagulation artifacts were mentioned in 1%.
According to ASCCP recommendations (15), the HSIL cytology is associated with cervical cancer in about 2%. In our study, all the invasive and micro invasive cancers diagnosed on the loop specimen had a HGSIL cytology, but the association was present in 3,34%.
All the patients with micro invasive or invasive results on the conisation specimen were under-evaluated for invasion on the pap smear (HGSIL) and at the colposcopy. An explanation could be that the lesion was not very large or it developed into the cervical canal. In the end, all these cases were cervical carcinoma 1A1, 1A2 or 1B1 of small volume, as we found only 2 invasive and 2 micro invasive lesions on the hysterectomy specimen after LLETZ.
On the other hand, in a region with a high incidence of cervical cancer like ours, Phongnarisorn(25) found in a LGSIL group (n=220), 36,4% HG lesions and 5% women with cancer, (9-microinvasive and 2 invasive carcinomas).
For the complication rate Zlatkov declare 7 hemorrhages (6.1%) and we had 2 cases (0.95%) who needed hospitalization. He encountered 8 histopathological results with artifacts of coagulation (19%) and we had 3.08%. (23) Mergui (24), in his study, reported the following complications: 2 cases with moderate cervical stenosis and 2 cases of postoperatory hemorrhagia treated by selective diathermocoagulation, similar to our results.

Conclusions
1. LLETZ and Fisher loop conisation effectuated at the end of a diagnostic algorithm for CIN lesions which includes the pap smear, the colposcopy and the HPV genotyping without punch biopsy, can be done with an acceptable rate of over-treatment, in our study it was 9.52%.
2. By this therapeutic attitude we can avoid the loose from the evidence of some cases between the time of biopsy and of conisation, and to under-evaluate some cases by punch biopsy.
3. In the same time the electrosurgical LLETZ/conisation is an efficient method for a conservatory treatment of CIN lesions, the rate of residual disease being low (14.76%).
4. The electrosurgical loop/conisation have a low rate of immediate or late complications (hemorrhagia, cervical stenosis) and it can be done in outpatient conditions under local anesthesia, and the alteration grade of the histopathological specimens by coagulation is low, so, from this point of view, the method could replace the cold knife conisation.
5. We are convinced that the HPV genotyping did not bring an important general increase in the sensibility and specificity of the combined examinations - pap smear and colposcopy - but its utility was evident in the follow up and in the diagnostic algorithm for the patients with unsatisfactory colposcopy.

References
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