Intraoperative HPV Testing Evaluation
Intraoperative HPV Testing Evaluation
High-grade intraepithelial lesion/cervical intraepithelial neoplasia grade 2-3 is a
 premalignant cervical lesion caused by persistent high-risk human papillomavirus infection.
 Human papillomavirus test is a very sensitive risk marker of cervical cancer and it has been
 incorporated in the follow-up after high-grade intraepithelial lesion treatment.
 Papillomavirus test performed intraoperatively could be a beneficial approach to anticipate
 treatment failure, allow for early management and consequently a reduction in costs. The aim
 of this study is to evaluate if the IOP-HPV test has non-inferior diagnostic utility of
 HSIL/CIN2-3 recurrence at 24 months as the HPV test performed 6 months after treatment.
Background:

 High-Grade Intraepithelial Lesion/Cervical Intraepithelial Neoplasia grade 2-3 (HSIL/CIN2-3)
 is a premalignant cervical lesion caused by persistent high-risk Human papillomavirus (HPV)
 infection [1]. It is estimated that approximately 54,000 women in Spain are annually
 diagnosed with HSIL/CIN2-3, which represents a significant economic burden for the national
 health system [2]. Electrosurgical excision procedure (LEEP) is the standard treatment of
 HSIL/CIN2-3 [3,4]. In Spain, current national guidelines recommend control after LEEP at 6,
 and 24 months [5,6,7]. The HPV test is a sensitive marker of cervical cancer risk and it has
 been incorporated, with cytology, in the follow-up of squamous cervical intraepithelial
 lesion after a LEEP [8,9,10].

 We recently showed that an HPV test performed intraoperatively (IOP-HPV) has a strong
 association with recurrent disease as well as a good diagnostic recurrence efficiency at 12
 months, similar than the test performed at 6 months. Thus, IOP-HPV test could be feasible and
 useful to identify treatment failure earlier than conventional strategies [11]. These results
 are similar to those previously observed by other authors [12].

 However, one of the limitations of previous studies was the sample size of patients to
 achieve greater statistical power and perform a non-inferiority study.

 The aim of this study is to evaluate if the IOP-HPV test has non-inferior diagnostic utility
 of HSIL/CIN2-3 recurrence at 24 months as the HPV test performed 6 months after LEEP.

 Material and methods

 This is a multicenter prospective cohort study that will include patients diagnosed with
 HSIL/CIN2-3. This study will be carried out at the Hospital Vall d'Hebron in Barcelona
 (organizing center) and other National centers from June 2020 to June 2024. All patients will
 undergo a HPV test 3 months prior to treatment and will be followed for a period of 24
 months.

 Cytology samples will be interpreted by an experienced pathologist following the Bethesda
 System [13]. HPV test will be performed using the commercially available Hybrid Capture 2
 (HC2) system. If this is positive, it will be followed by the CLART-HPV2 test, a PCR
 technique that will allow the detection of 35 HPV genotypes.

 Colposcopy will be performed using a colposcope Olympus 500 after preparing the cervix with
 5% acetic acid and lugol solution. Colposcopy findings will be described following the
 criteria of the International Federation for Cervical Pathology and Colposcopy (IFCPC) [14].

 Prior to LEEP, the abnormal area will be delimited by acetic acid and iodinated lugol, a
 colposcopy will be performed prior to the application of 1 ml of 2% mepivacaine to each
 quadrant of the cervix.

 The entire pathological area will be removed next to the transformation zone (TZ), followed
 by selective coagulation of the surgical bed by diathermic coagulation ball. Immediately
 after the LEEP, a cervical sample will be taken for the IOP-HPV test. At the end of the
 procedure an ECC will be performed using a Novak curette.

 The cervical specimen will be processed in a standardized way: after staining with ink the
 surfaces (or the margins) of the piece, a paraffin block will be obtained from which a
 minimum of 12 sections, of the 4 quadrants will be examined. A margin will be considered
 affected if the lesion reaches the margin or is within 1mm.

 After surgery, the follow-up will be performed according to the National Guidelines
 Recommendations5,6,7. Visits will be scheduled according to the margin status of the LEEP
 specimen: If the margins are negative, the patient will be reviewed at 6, 12 and 24 months
 with cytology, HPV test and colposcopy. If they are positive, a visit will be added 4 months
 after the procedure where an additional cervical cytology will be taken.

 Patients with abnormal cytology (LSIL+) or abnormal colposcopy will undergo a CGB. When the
 TZ is not completely visible or no colposcopic abnormalities are identified, an ECC with
 Novak curette will also be performed.

 High-grade recurrence will be considered either when the CGB confirmes HSIL/CIN2-3 or when
 the ECC shows HSIL.The criteria to perform a second treatment will be the histological
 confirmation of HSIL/CIN2-3 during the follow-up.
Inclusion Criteria:

 -  Diagnosis of HSIL/CIN2-3 by colposcopy guided biopsy (CGB) or endocervical curettage
 (ECC) in the 3 months prior to LEEP and/or confirmation of HSIL/CIN2-3 in the
 pathologic study of the surgical specimen

 Exclusion Criteria:

 -  Patients with acquired or congenital immunosuppression

 -  Patients undergoing chronic immunosuppressive treatments, prior treatment with LEEP

 -  Patients in whom HSIL/CIN2-3 was not histologically confirmed.