Implementation of HPV Testing in Patients After Radiotherapy for Cervical Cancer
Conditions: Cervical Cancer Recurrent, Radiotherapy, Cell Free DNA, HPV
Sponsor: General University Hospital, Prague

HPV After chemoRadioTherapy 
(HART) 
 
 
 
Protocol version 1.0 
Date 10.11.2025 
Sponsor General Faculty Hospital in Prague 
Principle investigator MUDr. Lukáš Dostálek, Ph.D. 
Full title of the trial Implementation of HPV Testing in Patients After 
Radiotherapy for Cervical Cancer 
Contact Apolinářská 18 
Praha 2, 12800 
Czech republic 
tel.: +420777980048 
lukas.dostalek@vfn.cz 
 
 

 
Research team 
 
Department of Gynaecology, 
Obstetrics and Neonatology, General 
Faculty Hospital in Prague 
MUDr. Lukáš Dostálek, Ph.D. 
MUDr. Adam Buryanek 
MUDr. Daniel Brynda 
prof. MUDr. Jiří Sláma, Ph.D. 
prof. MUDr. David Cibula, CSc. 
Department of Radiation Oncology, 
Bulovka University Hospital 
MUDr. Karolína Votavová 
Department of Oncology, General 
University Hospital in Prague 
MUDr. Věra Tomancová 
Institute of Medical Biochemistry 
and Laboratory Diagnostics -  
Laboratory of Oncogenetics, General 
University Hospital in Prague 
doc. MUDr. Petra Kleiblová, Ph.D. 
RNDr. Jana Soukupová, Ph.D.  
Ing. Nehasil Petr 
RNDr. Markéta Janatová, Ph.D. 
RNDr. Just Pavel, Ph.D. 
Bc. Olena Yakovchuk 
Institute of Medical Biochemistry 
and Laboratory Diagnostics -  STD 
Laboratory, General University 
Hospital in Prague 
Mgr Miroslava Slováčková 
prof. MUDr. Tomáš Zima, DrSc., MBA. 
 
 
 
 
 
HART - Protocol  v 1.0; 14.11.2025 
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Principal Investigator's Statement 
​
 
Full title: 
HPV After Radiotherapy 
Acronym: 
HART 
Protocol version: 
1.0 
 
Principal Investigator's 
Statement: 
By my signature below, I confirm that I will conduct the 
study in accordance with the protocol and in compliance 
with the accepted standards of Good Clinical Practice and 
the Declaration of Helsinki. I will adhere to all applicable 
laws and regulations governing the conduct of clinical 
studies. All information and data related to the study will be 
treated as confidential and will not be disclosed to third 
parties or used for any purpose other than the conduct of 
this study. 
 
 
 
 
 
 
 
 
 
Centre:​
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Department of Gynaecology, Obstetrics and 
Neonatology, General Faculty Hospital in 
Prague 
Principal investigator: 
 
MUDr. Lukáš Dostálek, PhD. 
Date: 
 
14.11.2025​
 
Signature:​
 
 
 
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Synopsis 
Acronyme 
HART 
Full title 
Implementation of HPV Testing in Patients After Radiotherapy for 
Cervical Cancer 
HART: HPV After chemoRadioTherapy 
Trial type 
Prospective multicenter observational 
Primary objectives 
Sensitivity of recurrence detection in patients after (chemo)radiotherapy 
(CRT) for cervical cancer during two-year follow-up using:  
1.​ Real-Time PCR of HPV DNA in a sample taken from a cervical 
swab 
2.​ Detection of HPV ctDNA from a patient's peripheral blood 
sample  
Sensitivities will be determined separately with respect to the occurrence 
of recurrence in the year following the sampling or throughout the entire 
two-year follow-up - see chapter Statistical Analysis for more details. 
Null hypotheses 
There is no difference in recurrence rates between patients who are 
HPV-positive vs. HPV-negative during follow-up after CRT for cervical 
cancer:  
1.​ using PCR of HPV DNA in a sample taken from a cervical swab 
2.​ using the detection of HPV ctDNA presence in a patient's 
peripheral blood sample 
 
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Secondary Objectives 
1.​ Rate of PCR HPV DNA negativity from a swab after CRT (at 
Visit 1)  
a.​ In case of HPV persistence, comparison of HPV 
genotype before and after completion of treatment  
2.​ Rate of HPV ctDNA negativity after CRT (at Visit 1) 
3.​ Comparison of the sensitivity of recurrence detection using PCR 
of HPV DNA in a sample taken from a cervical swab with the 
method of HPV ctDNA detection from a patient's peripheral 
blood sample  
4.​ Evaluation of the course of further treatment (especially the 
possibility of curability) in patients who were detected with 
recurrence during the study  
5.​ Analysis of HPV swab and cfDNA collection performed upon 
recurrence detection 
Porpose of the trial 
1.​ Implement HPV testing into clinical practice for patients after 
primary CRT for cervical cancer in the Czech Republic. 
2.​ Contribute to the introduction of the ctDNA monitoring method 
into clinical practice in the Czech Republic. 
3.​ Compare the prognostic significance of HPV positivity after CRT 
detected by HPV DNA PCR from a cervical swab with detection 
by determining the presence of HPV ctDNA from serum. 
4.​ Create a basis for prospective follow-up of patients who 
underwent a secondary surgical procedure based on the detection 
of recurrence in this study. 
Inclusion criteria 
1.​ Patient indicated for primary RT for cervical cancer 
2.​ FIGO stage IB - IVA 
3.​ Signed informed consent 
4.​ Age ≥ 18 years 
5.​ Administration of RT with curative intent 
Exclusion criteria 
1.​ Clinical stage FIGO IA 
2.​ Clinical stage FIGO IVB 
3.​ History of radiotherapy in the pelvis 
 
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4.​ Hysterectomy performed before the start of radiotherapy 
(adjuvant RT) 
5.​ History of HPV-associated malignancy in personal history 
6.​ HIV or other significant immunodeficiency 
Time schedule 
●​ duration: 4 years 
●​ study initiation: 1/2026 
●​ study completion: 1/2030 
Sample size 
120 patients 
 
 
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Contents 
Summary...................................................................................................................................................................9 
Follow-up of Patients after Primary CRT......................................................................................................... 9 
HPV PCR from a cervical swab......................................................................................................................10 
HPV PCR from a blood sample...................................................................................................................... 10 
Situation in the Czech Republic and Project Context..................................................................................... 11 
Study Objectives.....................................................................................................................................................11 
Secondary Study Objectives:.......................................................................................................................... 12 
Sample Size............................................................................................................................................................ 12 
Figure 1. The power of a one-sided test for N=60 - 150 with points corresponding to the studies cited in 
Tables 1 and 2 (alpha=0.05, one-sided)...........................................................................................................13 
Table 1. Background data for power analysis of the part of the study analyzing the implementation of a 
cervical swab...................................................................................................................................................13 
Table 2. Background data for power analysis of the part of the study analyzing the implementation of dPCR.
14 
Table 3. Power values of the study for individual sample sizes......................................................................15 
Study Procedure - SOP...........................................................................................................................................15 
Table 4. Visit schedule.....................................................................................................................................16 
Radiotherapy................................................................................................................................................... 16 
Course of Visits............................................................................................................................................... 18 
Imaging............................................................................................................................................................19 
Figure 2. Study scheme................................................................................................................................... 20 
Samples........................................................................................................................................................... 20 
Sending of Samples.........................................................................................................................................22 
Statistical Processing..............................................................................................................................................23 
Recurrence.......................................................................................................................................................23 
Evaluation of the Primary Objective...............................................................................................................24 
Exclusion of the Null Hypothesis....................................................................................................................24 
Evaluation of Secondary Objectives............................................................................................................... 24 
Figure 3. Statistical Processing....................................................................................................................... 25 
Data Processing...............................................................................................................................................25 
Information System................................................................................................................................................ 26 
Study Monitoring....................................................................................................................................................26 
Regulatory and Ethical Aspects of the Study.........................................................................................................26 
Financial Support of the Study...............................................................................................................................27 
Multicenter Collaboration...................................................................................................................................... 27 
Study Termination.................................................................................................................................................. 27 
Referrences.............................................................................................................................................................27 
 
 
 
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Abbreviations 
Abbreviation 
Full Name/Description (English) 
RT 
Radiotherapy 
CRT 
Chemoradiotherapy 
FIGO 
International Federation of Gynecology and Obstetrics 
HPV 
Human Papillomavirus 
SOP 
Standard Operating Procedure 
DFS 
Disease-free survival 
CR 
Complete Response 
SCC 
Squamous Cell Carcinoma Antigen 
ROC 
Receiver-operating Curve 
ddPCR 
Digital Droplet Polymerase Chain Reaction 
ctDNA 
circulating tumor DNA 
HPV-seq 
Sequencing of circulating HPV genome 
EBRT 
External Beam Radiotherapy 
BRT 
Brachyradiotherapy 
cfDNA 
Cell-free DNA 
CT 
Computed Tomography 
PET/CT 
Positron Emission Tomography/Computed Tomography 
MRI 
Magnetic resonance imaging 
PET/MRI 
Positron Emission Tomography/Magnetic resonance imaging 
D90 CTV HR 
Dose covering 90% of the target volume (High-Risk Clinical Target 
Volume) 
EQD2 
Biologically equivalent dose at 2 Gy fractionation (Equivalent Dose in 
2Gy Fractions) 
IMRT 
Intensity-Modulated Radiotherapy 
SIB 
Simultaneous Integrated Boost 
 
 
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Summary 
Human Papillomavirus infection is a causal factor for at least 95% of cervical cancers [1]. Surgical 
treatment and radiotherapy (RT) represent two fundamental modalities for the therapy of this disease, 
provided there is no distant metastasis. While surgical management is preferred in stage (FIGO) I, 
primary curative RT with concomitant chemotherapy (CRT) [2] plus targeted therapy with 
pembrolizumab is indicated in higher stages [3]. 
Follow-up of patients after surgical treatment for cervical cancer is codified by a number of 
recommended procedures and is based mainly on clinical examination, cytology collection, and HPV 
testing. The HPV test is mainly used in women who have retained their uterus after conservative 
surgery [4]. Especially in microinvasive carcinomas, the HPV test shows high sensitivity and a nearly 
100% negative predictive value. This means that its negativity almost excludes the future 
development of recurrence [5]. 
Follow-up of Patients after Primary CRT 
In patients after primary CRT, the uterus (including the cervix) is also preserved. However, the 
follow-up is governed by different principles. According to the EMBRACE-II protocol, therapeutic 
response in the tumor and lymph node area is assessed 3 months after completion of treatment using 
imaging (in Czech conditions, by performing PET/CT or PET/MRI) and gynecological examination. 
Regular follow-up is then started, which includes only gynecological examination. Imaging is 
repeated at 12 months after completion of treatment or at any time in case of suspected recurrence [6]. 
There is no consensus on the implementation of SCC antigen sampling into routine follow-up [2]. 
Implementation of imaging methods in later follow-up is not routinely recommended. Their indication 
is based on symptoms that lead to suspicion of recurrence [7]. For patients after CRT for cervical 
cancer, there is a clear recommendation not to collect a cervicovaginal cytology smear [2]. The 
implementation of the HPV test in the follow-up of these patients is not mentioned in current 
recommendations. 
Five-year disease-free survival (DFS) in patients after primary CRT is stage-dependent and ranges 
between 76% in stage IB and 47% in stage IVA [8]. Local (pelvic) recurrences in patients after CRT 
can be treated by surgical procedure with curative intent. Overall survival and quality of life then 
depend on the size of the recurrence, its location, and the extent of the surgery. However, even with 
highly complex procedures (extended exenteration), overall survival reaches almost fifty percent [9]. 
It follows from the above that the early detection of recurrence after primary CRT significantly 
increases the patient's chance of survival and quality of life. However, due to limited follow-up 
 
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options, recurrences are often not detected early in many cases, and patients can only be offered 
palliative treatment [10] 
HPV PCR from a cervical swab 
It has been repeatedly proven that the elimination of HPV after primary CRT (the so-called HPV 
clearance) represents one of the most fundamental positive prognostic factors arguing against the 
development of recurrence. Traditionally, achieving HPV clearance is determined using a swab from 
the preserved cervix [11–13]. In a significant proportion of patients, HPV DNA is not detectable in 
the cervical swab shortly after the completion of CRT. With increasing follow-up duration, the 
proportion of HPV-positive patients further decreases. In a 2011 study, the proportion of HPV-positive 
patients after one, three, and twelve months from the completion of treatment was 28%, 22%, and 
18%, respectively [11]. In this study, the development of recurrences (78%) was clearly associated 
with HPV persistence. The sensitivity and specificity of the HPV test taken from the cervix are 85% 
and 54%, respectively [12]. 
HPV PCR from a blood sample 
Another possibility for detecting persistence or recurrence is the detection of tumor DNA in the 
patient's circulation. Tumors associated with viral infection have long served as a model for 
developing methods for determining circulating tumor DNA (ctDNA) and for testing its clinical use 
[14]. 
HPV DNA is detected in the peripheral blood of patients with cervical cancer both at the time of 
diagnosis and at the relapse of the disease. Although it has been shown that HPV ctDNA levels reflect 
the probability of the presence of persistence or recurrence of an HPV-associated tumor, routine 
deployment has been hindered by low sensitivity. This was only 57% compared to PCR performed 
from a cervical swab [15]. 
In contrast, digital droplet PCR (ddPCR) brings a significant refinement to the detection of molecules 
at low concentrations [16]. ddPCR detected the presence of HPV ctDNA in 100% of patients with 
advanced cervical carcinoma before primary CRT. In an analysis performed after its completion, HPV 
ctDNA determination showed a sensitivity of 75% and a specificity of 80%. Furthermore, HPV 
ctDNA determination at three months after the completion of CRT showed higher accuracy compared 
to PET/CT in detecting recurrence after 18 months [17]. 
 
 
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Another possibility for increasing sensitivity is next-generation sequencing (NGS), whose sensitivity 
in determining circulating viral DNA (HPV-seq) surpasses the capabilities of ddPCR [18]. 
In a prospective clinical study examining the relationship between HPV ctDNA detection and the 
incidence of recurrences in patients after CRT for advanced cervical carcinoma, both methods were 
used—standard ddPCR and HPV-seq. It was again demonstrated that the detection of HPV ctDNA 
after the completion of CRT is associated with a poorer prognosis. Consistent with earlier work, an 
increase in sensitivity upon repeated 
Situation in the Czech Republic and Project Context 
In the Czech Republic (as in most other developed countries), HPV testing in the form of a simple 
cervicovaginal smear is currently not routinely performed for patients after CRT for cervical cancer. 
Also, monitoring of HPV ctDNA levels is limited to individual academic experiments, currently 
exclusively in the field of oropharyngeal carcinoma [22]. 
Testing for the presence of HPV DNA in the cervix and/or the patient's circulation can be an effective 
tool to identify a cohort of women who are at increased risk of recurrence *20*. These women can be 
subjected to more intensive monitoring for the development of recurrence, which should enable timely 
indication of surgical management or systemic treatment. Conversely, it is possible to define a group 
of negatively tested patients who should represent a cohort with an exceptionally good prognosis, in 
whom less intensive follow-up with a lower burden of imaging examinations could, conversely, be 
performed. 
The aim of this study is to document data that will enable the individualisation of follow-up based on 
HPV detection in patients after primary CRT for cervical cancer in the Czech Republic; to establish 
the relationship between the presence of HPV in the cervical smear or the patient's circulation and the 
occurrence of recurrence; to determine the rate of HPV infection elimination depending on the tumour 
characteristics and the administered treatment, and to evaluate treatment options for patients in whom 
recurrence was detected thanks to the study. 
Study Objectives 
Main Study Objectives: 
1.​ Sensitivity of recurrence detection using HPV DNA detection via Real Time PCR from a 
cervical swab in patients after (chemo)radiotherapy administered for cervical cancer 
 
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2.​ Sensitivity of recurrence detection using detection of HPV DNA fragments circulating in the 
peripheral blood of patients after (chemo)radiotherapy administered for cervical cancer 
Sensitivities will be determined separately with respect to the occurrence of recurrence in the year 
following the sampling or throughout the entire two-year follow-up - see the Statistical Analysis 
chapter for more details. 
Secondary Study Objectives: 
1.​ Rate of PCR HPV DNA negativity from a swab after CRT administration (comparison of 
HPV testing results within Screening with Visit 1) 
a.​ In case of HPV persistence, comparison of HPV genotype before the start of 
treatment and its completion 
2.​ Rate of HPV ctDNA negativity after CRT administration (comparison of HPV testing results 
within Screening with Visit 1) 
3.​ Comparison of the sensitivity of recurrence detection using PCR HPV DNA in a sample taken 
from a cervical swab with the method of HPV ctDNA detection from a patient's peripheral 
blood sample 
4.​ Evaluation of the course of further treatment (especially the possibility of curability) in 
patients who were detected with recurrence during the study 
5.​ Analysis of HPV swab and cfDNA collection performed upon the Unscheduled recurrence 
visit 
Sample Size 
The sample size calculation was performed by Mgr. Martin Komarc, PhD, Department of Biomedical 
Statistics (Biostat), 1st Faculty of Medicine, Charles University 
The significance of the difference in recurrence rates will be assessed using a one-sided test. To 
evaluate the effect size, graphs were constructed comparing Cohen's h with the power of the study for 
hypothetical cohorts of size 60 -150 patients (see Figure 1) 
 
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Figure 1. The power of a one-sided test for N=60 - 150 with points corresponding to the studies 
cited in Tables 1 and 2 (alpha=0.05, one-sided). 
 
Into these, the values of Cohen's h calculated from the studies listed in Tables 1 and 2 were entered 
based on specific initial proportions. The resulting powers for individual studies and the mentioned 
sample sizes are presented in Table 3. 
The power analysis was performed based on the following: 
Table 1. Background data for power analysis of the part of the study analyzing the 
implementation of a cervical swab 
HPV status after CRT 
Total 
Recurrence yes 
Recurrence no 
Song et al. [11] 
HPV negative 
127 
4 
123 
HPV positive 
29 
14 
15 
Total 
156 
18 (12%) 
138 
Nagai et al. [12] 
HPV negative 
42 
3 
39 
HPV positive 
50 
17 
33 
Total 
92 
20 (22%) 
72 
 
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Mahantshetty et al. [21] 
HPV negative 
43 
5 
38 
HPV positive 
89 
29 
60 
Total 
132 
34 (26%) 
98 
 
Table 2. Background data for power analysis of the part of the study analyzing the 
implementation of dPCR 
HPV ctDNA after CRT 
Total 
Recurrence yes 
Recurrence no 
Cabel et al [22] 
Nedetekována 
10 
2 
8 
Detekována 
4 
3 
1 
Total 
14 
5 (36%) 
9 
Han et al [17] 
Nedetekována 
13 
1 
12 
Detekována 
6 
3 
3 
Total 
19 
4 (21%) 
15 
Han et al [23] 
Nedetekována 
37 
9 
28 
Detekována 
25 
12 
13 
Total 
62 
21(34%) 
41 
 
Given that a power of ≥ 0.8 was achieved, the minimum sample size was determined to be 110 
patients (Table 3). After taking into account the anticipated study exclusion rate (drop-out rate), the 
target sample size was set at 120 patients. 
 
 
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Table 3. Power values of the study for individual sample sizes 
 
Cohen 
h 
Power for N= 
Study 
60 
70 
80 
90 
100 
110 
120 
130 
140 
150 
Song et al. 2011 
1,2 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
Nagai et al. 2004 
0,7 
0,7 
0,8 
0,8 
0,9 
0,9 
1,0 
1,0 
1,0 
1,0 
1,0 
Mahantsh. et al. 2018 
0,5 
0,5 
0,6 
0,6 
0,7 
0,7 
0,8 
0,8 
0,8 
0,9 
0,9 
Cabel et al. 2021 
1,2 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
Han et al. 2018 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
1,0 
Han et al. 2024 
0,5 
0,6 
0,6 
0,7 
0,7 
0,8 
0,8 
0,9 
0,9 
0,9 
0,9 
 
Study Procedure - SOP 
Patients who meet all inclusion criteria and none of the exclusion criteria may be enrolled. The study 
will proceed according to the scheme. 
 
 
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Table 4. Visit schedule 
 
Screening 
Visit 1 
Visit 2 
Visit 3 
Unscheduled 
recurrence visit 
Interval since 
CRT 
max 1 month 
before the start of 
CRT 
3 months 
 ± 14 days 1 
12 months 
 ± 30 days 
24 months 
 ± 30 days 
 
Informed 
consent 
+ 
 
 
 
 
Imaging 
 
+ 
+ 
+ 
+ 
HPV swab 
+ 
+ 
+ 
 
+/- 
ctDNA sample 
+ 
+ 
+ 
 
+/- 
SCC sample 
+/- 
+/- 
+/- 
+/- 
+/- 
Symptoms 
 
+ 
+ 
+ 
+ 
+ compulsory; +/- optional 
1In case of necessity to postpone the date of Visit 1 (for example, due to the coincidence of imaging with a visit 
in another study), the principal investigator must be consulted. 
 
Examinations indicated before the administration of radiotherapy are described below within the 
description of the Screening visit. 
Radiotherapy 
Radiotherapy must be indicated with curative intent. It must be primary radiotherapy for cervical 
carcinoma. It must not be adjuvant radiotherapy - the uterus must be left in situ. The indispensable 
standard treatment for locally advanced cervical carcinoma is external beam radiotherapy (EBRT) 
with a recommendation for concomitant chemotherapy and subsequent brachytherapy (BRT). Patients 
for whom only EBRT was indicated may also be included in the study. 
External Beam Radiotherapy to the pelvis, including pelvic and potentially para-aortic and inguinal 
lymph nodes, is a fundamental part of the treatment strategy with the main goal of achieving local and 
nodal control. The total dose of EBRT must be 45–50.4 Gy delivered in 23–28 fractions (1.8–2.0 Gy 
per fraction). Affected nodes will be treated with a Simultaneous Integrated Boost (SIB) so that the 
total dose (including the dose contribution from brachytherapy) reaches 60 Gy EQD2. Radiotherapy 
will be performed using one of the following techniques with daily image verification and potential 
correction of the patient's position: 
●​ IMRT (Intensity-Modulated Radiotherapy) 
●​ VMAT (Volumetric Modulated Arc Therapy) or 
 
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●​ Tomotherapy 
In case the patient is not indicated for brachytherapy or is unable to undergo it, an external beam 
radiotherapy boost will be performed to the maximum achievable dose, taking into account the 
tolerance of healthy tissues. 
Brachytherapy (if indicated) will be performed after the completion of external beam radiotherapy or 
during the 5th week of EBRT, with the aim of utilizing maximum tumor regression. BRT contouring 
and planning will be based on 3D image documentation obtained using CT or MRI. The total 
brachytherapy dose will be 25–30 Gy delivered in 4–6 fractions (5–7 Gy per fraction). To achieve 
adequate coverage of the target volume (CTV) and simultaneous adherence to dose limits for organs 
at risk (OAR), brachytherapy will be performed intracavitarily or as a combined intracavitary and 
interstitial technique. The sum of the EBRT and BRT doses will be evaluated using the calculation of 
the biologically equivalent dose at 2 Gy fractionation (EQD2), based on the linear-quadratic model 
with parameters α/β = 10 Gy for tumor effect and α/β = 3 Gy for late healthy tissue damage. The D90 
value for CTV-HR will be ≥ 85 Gy. 
Contouring of target volumes, organs at risk, and recommended fractionation will follow the 
EMBRACE II protocol [6]. The total treatment time, including both EBRT and BRT, should not 
exceed 56 days. Any interruptions of radiotherapy must be compensated according to local site 
standards. BRT administration is not a mandatory condition for study enrollment. 
Chemotherapy (if indicated) will be administered concurrently with external beam radiotherapy 
(concomitantly). The treatment regimen includes cisplatin at a dose of 40 mg/m² administered once a 
week, always on the day of planned EBRT. In case of treatment prolongation and the need to continue 
chemotherapy after the completion of EBRT, cisplatin will not be administered on the days of 
brachytherapy application. The goal is to administer 5–6 cycles of concomitant chemotherapy. Dose 
reduction, reduction in the number of cycles, or complete omission of chemotherapy due to toxicity or 
contraindications on the patient's side is permissible and will be managed by the treating physician in 
accordance with local standards of care. 
Concomitant and Adjuvant Immunotherapy - if the criteria of the KEYNOTE-A18 / ENGOT-cx11 
study [3] are met, pembrolizumab may be administered concomitantly and subsequently as adjuvant 
therapy according to the recommended dosing. The management of administration and toxicity 
treatment will be led by the treating physician in accordance with current international and local 
recommendations. 
 
 
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Course of Visits 
●​ During the Screening visit, which will take place before the start of CRT, the study will be 
thoroughly explained to the patient and informed consent will be signed. The visit must not 
precede the start of CRT by more than 1 month. Furthermore, an HPV DNA test will be taken 
from the cervix and a blood sample will be collected to determine the presence of ctDNA 
HPV. Patient characteristics, tumor properties, and the result of clinical staging (FIGO) will 
be recorded in the eCRF. 
●​ Visit 1 will follow 3 months after the completion of CRT ± 14 days. (In case of necessity to 
postpone the date of Visit 1 (for example, due to the coincidence of imaging with a visit in 
another study), the principal investigator must be consulted.) Imaging will be performed 
during this visit. Furthermore, an HPV swab and a blood sample for ctDNA determination 
will be collected. The SCC marker may also be collected during Visit 1. Its value will be 
determined locally and the result will be recorded in the eCRF. Any symptoms of the patient 
will also be recorded. 
●​ Visit 2 will follow 12 months after the completion of CRT ± 30 days. Imaging will be 
performed during this visit. Furthermore, an HPV swab and a blood sample for ctDNA 
determination will be collected. The SCC marker may be collected during Visit 2. Its value 
will be determined locally and the result will be recorded in the eCRF. Any symptoms of the 
patient will also be recorded. 
●​ Visit 3 will follow 24 months after the completion of CRT ± 30 days. Imaging will be 
performed during Visit 3. The SCC marker may also be collected. Its value will be determined 
locally and the result will be recorded in the eCRF. Any symptoms of the patient will also be 
recorded. 
●​ In case of recurrence detection, an Unscheduled Recurrence Visit will be performed. Imaging 
will be performed during this visit. During this visit, an HPV swab and a blood sample for 
ctDNA determination will be collected. The SCC marker may also be collected. Its value will 
be determined locally and the result will be recorded in the eCRF. Any symptoms of the 
patient will also be recorded. This visit will also be completed if recurrence is detected 
outside of Visits 2 and 3 (for example, by performing imaging for other reasons). 
Treatment of a potential recurrence depends on the decision of the multidisciplinary 
oncogynecological board. The patient will be further monitored in the prospective study registry. The 
type of recurrence (local, combined, distant), location of recurrence, proposed treatment, and the 
result of this treatment (CR vs. non-CR) will be recorded. 
 
 
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In case the patient does not want to or cannot continue (for example, due to withdrawal of consent, 
death, etc.), the EoT Visit will be completed. 
Imaging 
PET/CT or PET/MRI is accepted as imaging. In case only CT or MRI is performed, the principal 
investigator will be contacted, who will decide on the possibility of accepting this imaging. The 
reading will be performed locally. Imaging performed during Visit 1 will serve for the assessment of 
therapeutic response to CRT and the achievement of CR. Imaging performed during Visits 2, 3, and 
during the Unscheduled Recurrence Visit will serve to identify patients with recurrence - see the 
chapter.
 
 
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Figure 2. Study scheme 
 
Samples 
The Evalyn self-sampling set (ROVERS MEDICAL DEVICES, Lekstraat 10, NL-5347 KV Oss., 
Netherlands) will be used to perform the cervical swab. The sets will be distributed in sufficient 
numbers to individual workplaces. The test will be taken by the examining clinician according to the 
instructions, or the patient may take it herself (the method of collection will be recorded in the eCRF). 
After collection, the set will be sent according to the instructions below. 
 
Full genotyping of all 14 high-risk HPV genotypes will be performed. 
 
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Venous blood will be drawn into a tube intended for ctDNA/cfDNA collection and preservation (e.g., 
Streck Cell-Free DNA BCT, PAXgene Blood ccfDNA Tube, AmoyDx® Cell-free DNA Protection 
Vacuum Tube). The sample will be processed according to the manufacturer's protocol. DNA isolation 
will be performed manually from 10 ml of blood for one examination using the QIAamp MinElute 
cfDNA Midi Kit (Qiagen, (Qiagen, QIAGEN Straße 1, 40724 Hilden, Germany), quantified and 
stored at -80°C until subsequent processing. 
cfDNA analysis will be performed using panel NGS with target enrichment technology utilizing a 
custom panel targeting high-risk HPV genomic DNA and selected human ctDNA targets. Library 
preparation will be done using the KAPA EvoPrep Kit (Roche) with UMI adapters. Sequencing will 
take place on the Illumina platform (NexSeq/NovaSeq; 5200 Illumina Way, San Diego, CA 92122 
USA). Bioinformatics processing will be performed using an optimized in-house bioinformatics 
pipeline (Laboratory of Oncogenetics) 
 
 
 
 
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Sending of Samples 
1.​ Envelopes for sending samples will be distributed to individual centers. Please send the 
samples immediately after collection (the same day)! Procedure for sending samples: 
2.​ Mark the samples (Evalyn brush and tube/tubes for cfDNA) with the patient identifier - use 
the same one you selected for entry in RadCap - CENTERNAME_XXX 
3.​ Place the samples in the envelope 
4.​ Order transportation at DHL: 
+420 220 300 111 
5.​ Use following details: 
account number: 
304117963 
owner name: 
Všeobecná fakultní nemocnice v Praze 
 
Address for sending: 
Adam Buryanek 
Klinika gynekologie, porodnictví a neonatologie 
Apolinářská 18 
Praha 2 
128 00 
Czech republic 
 
The results of the HPV testing will be provided to the attending physician. Further procedure will be 
under the sole responsibility of the attending physician. 
 
In case of urgent issues/clarifications, please call the principal investigator: 
Lukáš Dostálek 
+420 777 98 00 48 
 
 
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Statistical Processing 
Recurrence 
Recurrences will be evaluated in patients who achieved CR after the completion of CRT. 
Recurrence is defined as a clear evidence of avid tissue on PET/CT or PET/MRI (oncological 
probability scale = 5 - positive finding). In case of equivocal findings, recurrence must be verified by 
biopsy. 
Recurrence may be detected in the following ways: 
●​ by imaging indicated within Visit 2 or 3 
○​ (recurrence detected within Visit 1 will be labelled as persistence or non-CR) 
●​ by imaging indicated outside the prescribed visits - "Unscheduled Recurrence Visit"; 
○​ *Note: in case of recurrence detection outside the study but within the monitored 
follow-up (two years from the completion of CRT) (for example, during imaging 
indicated at another workplace, etc.), it is ***always*** necessary to complete the 
“Unscheduled Recurrence Visit”* 
○​ *If in this case PET/CT or PET/MRI was not performed as imaging and its result is 
clear (i.e., the treating physician does not indicate a supplementary PET/CT or 
PET/MRI), the principal investigator (MUDr. Lukáš Dostálek, Ph.D.) will be 
contacted, who will decide on further procedure* 
Recurrences will be defined by temporal distribution as follows: 
●​ Recurrence in the first year will be labelled as the one that occurs between Visit 1 and 
imaging indicated within Visit 2 inclusive 
●​ Recurrence in the second year will be labelled as the one that occurs between Visit 2 and 
imaging indicated within Visit 3 inclusive; this applies to patients who did not experience 
recurrence in the first year 
●​ Recurrence during the entire follow-up will be labelled as the one that occurs between Visit 1 
and imaging indicated within Visit 3 inclusive 
Recurrences will be defined by localization as follows: 
●​ local, affecting only the cervix, including those that spread continuously to its surroundings 
●​ regional, where the tumor occurs in the pelvis without connection to the cervix 
●​ distant, where the tumor occurs outside the pelvis and does not occur in the cervix 
●​ combined - a combination of local and distnat. 
 
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Evaluation of the Primary Objective 
Only patients with confirmed CR within Visit 1 will be included in the evaluation of the primary 
objective (sensitivity). 
Sensitivity in the periods defined below will be calculated as follows (see Figure 
 
𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐻𝑃𝑉 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑖𝑛 𝑤ℎ𝑜𝑚 𝑟𝑒𝑐𝑢𝑟𝑟𝑒𝑛𝑐𝑒 𝑜𝑐𝑐𝑢𝑟𝑟𝑒𝑑
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑖𝑛 𝑤ℎ𝑜𝑚 𝑟𝑒𝑐𝑢𝑟𝑟𝑒𝑛𝑐𝑒 𝑜𝑐𝑐𝑢𝑟𝑟𝑒𝑑
Sensitivity will be calculated separately as: 
●​ Sensitivity of the entire follow-up - patients who tested HPV positive at Visit 1 related to 
recurrences detected throughout the entire follow-up. 
●​ Sensitivity of the 1st year - patients who tested HPV positive at Visit 1 related to recurrences 
detected during the first year. 
●​ Sensitivity of the 2nd year - patients who tested HPV positive at Visit 1 or Visit 2 related to 
recurrences detected during the second year. 
●​ Sensitivities will be calculated separately in this way for HPV testing using a cervicovaginal 
swab and ctDNA. 
Exclusion of the Null Hypothesis 
The prognosis of HPV positive patients (by both tests) and HPV negative patients will be plotted 
using the Kaplan-Meier curve. Differences in prognosis (HPV positive vs. HPV negative) will be 
evaluated using the log-rank test. 
Evaluation of Secondary Objectives 
The rate of negativity will be evaluated separately for HPV testing using a cervicovaginal swab and 
ctDNA. It will be calculated as follows: 
 
1 − 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐻𝑃𝑉 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑤𝑖𝑡ℎ𝑖𝑛 𝑡ℎ𝑒 𝑆𝑐𝑟𝑒𝑒𝑛𝑖𝑛𝑔 𝑣𝑖𝑠𝑖𝑡
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐻𝑃𝑉 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑤𝑖𝑡ℎ𝑖𝑛 𝑉𝑖𝑠𝑖𝑡 1
Furthermore, the relationship between the negativity of both tests and the achievement of CR on 
imaging within Visit 1 will be evaluated. This analysis will be performed using Chi-squared test. 
Comparison of the sensitivities (and other parameters such as specificity, negative predictive value, 
etc.) of both tests will be performed using analysis of the ROC curve. 
 
 
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Figure 3. Statistical Processing 
 
Data Processing 
The parameters obtained by this analysis (sensitivity, specificity) will be plotted on an ROC curve. 
The deviation of the ROC curve from the level of chance (AUC = 0.5) will be evaluated using the 
Z-test according to the method of Hanley and McNeil (1982). Other parameters will be processed 
using standard methods of descriptive statistics, including relative and absolute frequencies and 
arithmetic mean with standard deviation. The chi quare test will be used to compare the frequencies of 
demographic and clinical categorical variables. Continuous variables will be compared using the 
Mann-Whitney U test, which is a non-parametric alternative to the t-test for independent samples. The 
level of statistical significance will be set for all tests at alpha = 0.05 
 
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Information System 
Data will be entered in pseudonymized form into an electronic database via eCRF. For this purpose, 
REDCap implemented under the 1st Faculty of Medicine of Charles University. 
 
redcap.lf1.cuni.cz 
 
Patients will be pseudonymized in the system - they will be entered into RedCap using identifiers 
chosen by each center. The recommended format is: CENTERNAME_XXX. Each center will 
maintain a table from which it will be possible to identify a specific patient by the identifier (patient 
log). 
Study Monitoring 
Oversight of the study conduct will be ensured by a monitor who will check compliance with basic 
legal norms and the quality of the collected data. Within the scope of centralized monitoring, they will 
supervise whether the rights and health of study subjects are protected, whether the recorded data is 
correct, complete, and verifiable, and whether the clinical study is being conducted in accordance with 
the latest approved version of the Protocol/amendments, Good Clinical Practice, and legal regulations. 
Regulatory and Ethical Aspects of the Study 
Participating sites are responsible for obtaining local ethical committee approval. Each patient will 
voluntarily agree to participate in the clinical trial and will sign an informed consent form before 
being enrolled in the study. The study will be conducted in accordance with the protocol terms and 
generally accepted standards of good clinical practice, in compliance with the guidelines of the 
International Conference on Harmonisation of Good Clinical Practice (ICH-GCP) and the Declaration 
of Helsinki. Investigators will comply with all applicable laws and regulations governing the conduct 
of clinical trials. Investigators will treat all information and data related to the study as confidential 
and will not disclose this information to any third parties or use it for any purpose other than the 
conduct of this study. 
 
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Financial Support of the Study 
Institutional support from the General Faculty Hospital will be requested in 2026. 
Multicenter Collaboration 
The treatment of each patient is led in accordance with local standards. The local physician or 
principal investigator must explain the nature of the study and the associated procedures to the patient 
in detail. The local physician or principal investigator bears responsibility for any complications 
arising from the treatment, including radiotherapy toxicity. Users will have access to the REDCap 
system via major web browsers without the need to install any additional software. Local investigators 
will receive a username and password to access the REDCap system. Communication between the 
server and users will be secured by an encrypted protocol (HTTPS) and Secure Socket Layer 
encryption. Only pseudonymized data will be collected; local investigators will create a local 
identifier for local patient identification. 
Study Termination 
The study will end with the completion of Visit 5 for the last monitored patient. 
 
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