Tru-cut Biopsy in Tumor Characterisation
Conditions: Tumor
Sponsor: General University Hospital, Prague

TRUTH – TRU-cut biopsy in Tumor cHaracterisation 
 
 
 
 
 
 
 
 
 
 
Version:  
 
2.0 
Date:   
 
1-JAN-2024 
Sponsor:  
 
General University Hospital in Prague 
Main investigator:  Renata Poncova 
Co-investigator:  
Filip Frühauf 
 
Contact address:  
Apolinarska 18 
Prague 2, 128 51 
Czech Republic 
tel.: +420224967451 
e-mail: renata.poncova@vfn.cz 
Trial chairs:  
Renata Poncova 
 
 
 
Filip Frühauf 
David Cibula 
Biostatistician:  
Jiri Jarkovsky 
Study coordinator: Dominika Prihodova 
Study design: 
Prospective cohort 
 

2 
DECLARATION OF THE MAIN INVESTIGATOR  
  
Title: A single arm, prospective, cohort study to evaluate the feasibility of a complex classification of 
pelvic and abdominal tumors by tru-cut biopsy with immunohistochemistry, next-generation 
sequencing, and immunological examination. 
   
Acronym: TRUTH 
  
Protocol version: 2.0  
The undersigned confirms that the above-referenced protocol has been acknowledged and accepted, 
he agrees to conduct the study in compliance with the approved protocol, and will adhere to the ICH 
guidelines, the most recent version of the Declaration of Helsinki, the EU General Data Protection 
Regulation 2016/679 (GDPR), relevant Czech laws implementing the GDPR, and any other regulatory 
requirements and Standard Operating Procedures (SOPs), as applicable. 
 
The undersigned agrees not to disclose the confidential information contained in this document for 
any purpose other than the evaluation or conduct of the study, without prior written consent from the 
sponsor. 
  
Center: Department of Obstetrics and Gynecology 
  General University Hospital and First Faculty of Medicine Charles University  
  Prague  
  
Main investigator: Renata Poncová 
  
Date: 1-JAN-2024 
  
Signature:
 
  
  
 
 
 
 
 
 
 

3 
SYNOPSIS 
 
Title 
A prospective study to evaluate the feasibility of a complex classification of pelvic 
and abdominal tumors by tru-cut biopsy with immunohistochemistry, next-
generation sequencing, and immunological examination. 
Acronym 
TRUTH  
Type 
Prospective cohort 
Objective 
The objective of this prospective study is to evaluate if a complex classification of 
pelvic and abdominal tumors using tissue samples obtained by tru-cut biopsy 
(TCB) is feasible. 
Primary endpoint 
The primary endpoint is the adequacy of samples obtained by TCB for 
histopathological examination. 
Secondary 
endpoint 
The secondary endpoints are the adequacy of samples obtained by TCB for: 
1. next-generation sequencing examination 
2. immunohistochemistry examination 
3. immunologic examination 
Additional 
endpoints 
1. To evaluate which factors affect the primary and secondary endpoints, 
including: 
a. tumor origin 
b. size of the tumor 
c. presence of ascites 
d. body mass index (BMI) 
e. biopsy site 
f. 
biopsy approach (transvaginal, transrectal vs. transabdominal) 
g. disease status – first diagnosis, recurrence or progression 
h. previous treatment modality – chemotherapy, anti-angiogenic 
therapy, PARP inhibitor therapy, hormonal therapy, or radiotherapy 
2. To evaluate the overall safety of TCB 
3. To evaluate the overall accuracy of TCB for complex classification of pelvic 
and abdominal tumors 
Inclusion criteria 
1. Presence of a pelvic/abdominal lesion with suspicion of malignancy: 
a. new diagnosis of a presumed gynecologic tumor 
b. suspicious recurrence or progression of a known gynecologic 
malignancy 
c. pelvic/abdominal spread or recurrence of a non-gynecologic tumor 
(primary tumor or metastasis) 
d. pelvic/abdominal spread of a tumor of unknown origin 
2. Age ≥18 years 
3. Eastern Cooperative Oncology Group (ECOG) performance status grade <3 
4. Not pregnant 

4 
5. Signed informed consent form 
Exclusion criteria 
1. Considered by the investigator to be unsuitable for any treatment 
2. Early stage disease 
3. Age <18 years 
Time frame and 
sample size 
A total of 250 consecutive patients 
Start date: 1-FEB-2024 
End date: 31-JAN-2026 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

5 
TABLE OF CONTENTS  
  
DECLARATION OF THE MAIN INVESTIGATOR  
 
 
 
 
2 
SYNOPSIS 
 
 
 
 
 
 
 
 
 
 
3 
TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
5 
LIST OF ABBREVIATIONS  
 
 
 
 
 
 
 
6 
ROLES AND RESPONSIBILITIES  
 
 
 
 
 
 
7 
1. BACKGROUND AND RATIONALE 
 
 
 
 
 
 
8 
2. ENDPOINTS 
 
 
 
 
 
 
 
 
 
9 
3. INCLUSION CRITERIA  
 
 
 
 
 
 
 
10 
4. EXCLUSION CRITERIA  
 
 
 
 
 
 
 
10 
5. STUDY DESCRIPTION  
 
 
 
 
 
 
 
11 
6. STATISTICAL ANALYSIS 
 
 
 
 
 
 
 
18 
7. STUDY TIMELINE AND SAMPLE SIZE  
 
 
 
 
 
18 
8. EXPECTED OUTCOMES 
 
 
 
 
 
 
 
19 
9. ETHICAL COMMITTEE APROVAL 
 
 
 
 
 
 
19 
10. DATA HANDLING AND MONITORING  
 
 
 
 
 
20 
11. FUNDING AND SUPPORT 
 
 
 
 
 
 
 
20 
12. PUBLICATION PLAN 
 
 
 
 
 
 
 
 
20 
13. REFERENCES 
 
 
 
 
 
 
 
 
 
21 
APPENDICES 
 
 
 
 
 
 
 
 
 
23 
 
 

6 
LIST OF ABBREVIATIONS 
 
ADNEX  
Assessment of Different NEoplasias in the adneXa (model) 
AE  
 
Adverse Events 
BMI 
 
Body Mass Index 
BOT 
 
Border-line Ovarian Tumor 
CT 
 
Computed Tomography 
ECOG PS 
Eastern Cooperative Oncology Group Performance Status 
EC 
 
Ethical Committee 
FNAB  
Fine Needle Aspiration Biopsy 
GCP  
Good Clinical Practice 
HGSC  
High Grade Serous Carcinoma 
ICF 
 
Informed Consent Form 
IHC 
 
Immunohistochemistry 
ICH 
 
International Conference on Harmonisation 
LGSC   
Low Grade Serous Carcinoma 
LMWH  
Low Molecular Weight Heparin 
MI 
 
Main Investigator 
MRI 
 
Magnetic Resonance Imaging 
NACT   
Neoadjuvant Chemotherapy 
NGS   
Next-Generation Sequencing 
NOAC  
New Anticoagulants 
PI 
 
Principal Investigator 
PARP   
Poly-ADP Ribose Polymerase 
PET 
 
Positron Emission Tomography 
REDcap  
Research Electronic Data Capture 
TAS 
 
Transabdominal Ultrasound 
TCB   
Tru-cut Biopsy 
TIL  
 
Tumor Infiltrating Lymphocytes 
TMB 
 
Tumor Mutational Burden 
TVS 
 
Transvaginal Ultrasound 
US 
 
Ultrasound 
 
 
 
 
 
 

7 
ROLES AND RESPONSIBILITIES 
 
The principal investigator (PI) is responsible for the conduct of the study at his/her participating site, 
and for protecting the rights, safety, and well-being of all participants. As such, the PI must ensure 
adequate supervision of the study at the local site. If any tasks are delegated, the PI will maintain a 
log of appropriately qualified persons to whom he/she has delegated the specified study-related 
duties. The PI will ensure that adequate training is provided and documented for all study staff, before 
they conduct the assigned activities. Even when certain activities are delegated, the PI will ultimately 
remain responsible for the conduct of the study. 
The main investigator’s (MI) responsibility is to supervise the general conduct of the study, 
including study progress, communications, protocol training and support of the participating sites, 
annual reporting to the ethical committee (EC), end of study notification(s), and reporting of the 
results. The MI fulfils both investigator and sponsor responsibilities, as outlined in the International 
Conference on Harmonisation – good clinical practice (ICH-GCP) and applicable regulations. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

8 
 
1. BACKGROUND AND RATIONALE 
 
Image-guided diagnostic interventions are valuable tools in gynecologic oncology clinical practice. 
Several procedures are available, including core needle (tru-cut) biopsy (TCB) and fine needle 
aspiration biopsy (FNAB).  
The purpose of performing biopsies in gynecologic practice is to obtain a representative tissue 
sample for a valid morphological examination. Biopsies are particularly useful for patients who are 
deemed unsuitable for more invasive interventions, and accelerate the process from diagnosis to 
therapy without the need for a period of recovery [1, 2]. In addition, in the era of personalized medicine, 
biopsies of gynecological cancers, either from primary, recurrent or progressive disease, have 
become important for characterizing the cellular pathways of the disease and to plan subsequent 
targeted therapy [3]. 
Tru-cut biopsy (TCB) is a minimally invasive method to obtain an adequate sample from the 
lesion and hence tailor the subsequent management avoiding unnecessary surgery. In gynecologic 
oncology, this technique is predominantly used in advanced ovarian cancers if primary cytoreductive 
surgery with no residual disease is not feasible and for patients indicated for neoadjuvant 
chemotherapy. Furthermore, it is useful to verify recurrent disease [4] and can be used in cases with 
tumors of presumed non-gynecologic origin that, in the pelvis, are mostly metastases derived from 
the gastrointestinal tract or breast cancer [5]. TCB can be used in combination with subjective 
ultrasound (US) assessment or with the assessment of different neoplasias in the adnexa (ADNEX) 
model to evaluate the nature of the majority of the pelvic tumor [6]. 
The Gynecologic Oncology Centre of the General University Hospital in Prague was among 
the first centers to start using TCB. Also, one of the largest retrospective studies published on this 
topic was done in our center. In the previous study, 190 patients were enrolled, and an adequate 
sample was obtained in more than 93% of all cases. In patients indicated for primary surgery or 
secondary debulking surgery, less than 2% of tru-cut biopsies did not coincide with the final histologic 
diagnoses. Regarding safety, only two bleeding complications, which were resolved by surgery, were 
recorded [7]. 
The samples obtained by TCB using an appropriate thick needle is usually sufficient for 
histopathological examination, immunohistochemistry (IHC), and next-generation sequencing (NGS). 
Although there is not yet strong evidence to show whether thick needle specimens are sufficient for 
the assessment of the tumor microenvironment based on immune monitoring, the available data are 
promising [8]. By contrast, FNAB only permits cytological examination with several crucial limitations 
to establish the final diagnosis [9, 10]. 

9 
Modern examination methods (IHC, NGS, immunology) can give new information about the 
tumor’s characteristics that can substantially affect the clinical prognosis and efficacy of different types 
of targeted treatment. Now, de novo biopsy in cases of disease recurrence or progression is strongly 
recommended because it can reveal the alterations of these characteristics that occur during the 
course of treatment and improve the therapeutic strategies. In ovarian cancer, high temporal 
heterogeneity of tumor biopsies has been demonstrated, with a significant clinical impact [11]. 
TCB can be considered the smartest way to histopathologically verify a gynecologic 
malignancy because it is minimally invasive, is performed in an outpatient basis, and is well tolerable 
with a low complication rate. Furthermore, TCB has an excellent cost‒benefit ratio. 
 
2. ENDPOINTS 
 
2.1. Primary endpoint 
The primary endpoint is the adequacy of samples obtained by TCB for histopathological diagnosis at 
the Gynecologic Oncology Center of the General University Hospital in 3 years. 
 
2.2. Secondary endpoint 
The secondary endpoint is the adequacy of samples obtained by TCB for next generation sequencing, 
immunohistochemistry, and immunologic examination. 
 
2.3. Additional endpoints 
 
1. To evaluate which factors affect the primary and secondary endpoint, including: 
a. tumor origin 
b. size of the tumor 
c. presence of ascites 
d. body mass index (BMI) 
e. biopsy site 
f. biopsy approach transvaginal, transrectal vs. transabdominal 
g. disease status – the first diagnosis, the first and other recurrence or progression 
h. previous treatment modality – chemotherapy, anti-angiogenic therapy, PARP inhibitor 
therapy, hormonal therapy, or radiotherapy 
2. To evaluate the overall safety of TCB 
3. To evaluate the overall accuracy of TCB for complex classification of pelvic and abdominal tumors 
 
 
 

10 
2.4. Definitions of endpoints 
 
The feasibility of TCB will be calculated as the proportion of patients in whom TCB was performed 
among all patients enrolled in the study.  
Adequacy is defined as the proportion of patients in whom an adequate sample was obtained 
for a particular examination from among those patients who underwent TCB.  
Accuracy will only be evaluated in patients in whom an adequate sample was obtained by TCB 
who underwent surgery with biopsy. Accuracy is defined as the concordance between the 
examination of samples obtained by TCB and the results of surgery with TCB to the final 
histopathology. The outcome of the final histopathological examination will be considered the 
reference standard.  
For accuracy and adequacy, the impact of selected variables (e.g., approach, number of 
biopsies, and target lesion size) will be assessed in a multivariable analysis. 
Safety is defined as the rate of complications related to TCB, including events occurring during 
the procedure, events within 24 hours after the procedure (early events), and events within 30 days 
of the procedure (late events). 
 
3. ELIGIBILITY CRITERIA 
3.1. 
Inclusion criteria 
Patients will be eligible for the study if they satisfy all of the following criteria: 
1. Presence of a pelvic/abdominal lesion with suspicion of a primary malignancy, disease 
recurrence, or disease progression 
a. new diagnosis of a presumed gynecologic tumor 
b. suspected recurrence or progression of a known gynecologic malignancy 
c. pelvic/abdominal spread or recurrence of a non-gynecologic tumor (tumor itself or 
metastasis) 
d. pelvic/abdominal spread of a tumor of unknown origin. 
2. Age ≥18 years 
3. Eastern Cooperative Oncology Group Performance Status (ECOG PS) grade <3 
4. Not pregnant 
5. Signed informed consent form 
3.2. 
Exclusion criteria 
Patients will be excluded from the study for the following reasons: 
1. Considered by the investigator to be unsuitable for any treatment 
2. Early stage disease 
3. Age <18 years 

11 
4. STUDY DESCRIPTION 
 
Patients referred to the Gynecologic Oncology Centre with a new or recurrent progressive 
abdominal/pelvic tumor will be clinically examined. The standard of care imaging method will be 
performed as part of preoperative work-up, including expert transvaginal US (TVS), transrectal and 
transabdominal US (TAS) or computed tomography (CT), magnetic resonance imaging (MRI), or 
positron emission tomography (PET)/CT scan. 
Eligible women meeting all the inclusion criteria will be identified by a clinician. They will be 
provided with the “Informed consent” and “Information for patients” documents, and the objectives 
and the course of the study will be discussed with them. After obtaining written consent, patients will 
be registered into the database using the REDCap (Research Electronic Data Capture, www.project-
redcap.org) electronic data capture tool. This is a secure, web-based application with controlled 
access designed to support data capture for research studies, hosted at MD Anderson. Basic clinical 
data about patients will be compiled using the Clinical data form. The REDCap service complies with 
GDPR/data regulations. 
Before the procedure, the patient’s risk factors for bleeding (e.g. history of bleeding, and 
current use of antiplatelet or anticoagulant agents) will be carefully evaluated. In patients with any risk 
factors, their platelet (PLT) count and coagulation status will be tested and controlled by the physician 
performing TCB. In patients with thrombocytopenia or a prolonged prothrombin time and activated 
thromboplastin time, preventive measures should be discussed with a hemostasis specialist. PLT 
count > 50 × 109/L and an international normalized ratio (INR) > 1.5 are considered safe [12, 13]. In 
patients using low molecular weight heparin (LMWH), TCB should be not be performed within 12 h 
after the loading dose, or within 24 h after a therapeutic dose. In patients using vitamin K antagonists, 
TCB should be performed at least 5 days after last tablet [14]. In patients using new anticoagulant 
drugs (NOAC), it is only necessary to skip a single dose before and after TCB. Patients who are 
unable to undergo TCB due to hematologic complications will be excluded from the trial. 
 
4.1. DESCRIPTION OF TCB 
 
Because of the very low rate of complications associated with pelvic TCB, it is considered a low-risk 
procedure and, as such, can be performed in an outpatient basis. It does not require any special 
preparation of the patient, laboratory tests (except in patients with high risk of bleeding), fasting, or 
general anesthesia, and it involves a short procedure time. Anxious, risky patients and those who are 
unable to tolerate US should be offered conscious sedation or general anesthesia in an inpatient 
setting. Patients referred for surgical treatment according to clinical staging will undergo TCB just 

12 
before main surgery under anesthesia. For the other patients, TCB should be done as soon as 
possible after US, with a maximum interval of 1 week. 
The physician who performs TCB according to US will choose the best suitable approach, 
such as vaginal/ transrectal or abdominal/percutaneous; in some cases, both routes may be used. If 
the lesion is accessible by two different approaches (transvaginal and percutaneous), the preferred 
approach is transvaginal because the needle is easier to control, the needle tip can be continuously 
monitored easily, the higher resolution of the endovaginal probe, and the gynecologist usually has 
greater experience of this approach. All biopsies will be performed by physicians with training in 
performing gynecologic US and TCB.  
Antibiotic prophylaxis is recommended after transrectal biopsy if the needle passes through 
the rectal wall into the peritoneal cavity and in immunocompromised patients [15, 16]. The main 
principles of clean contaminated procedures must be followed when performing invasive procedures 
to minimize the risk of infection. 
 
4.1.1. Transvaginal TCB 
 
The patient will be placed in the lithotomic position in an US room or operating theatre. The needle 
will be inserted into an automatic biopsy gun. For the transvaginal approach, an 18G needle with a 
length of 30 cm will be used. The sample length may be 15 or 22 mm, and the required length of 
sample will be set. After disinfecting the vagina, the endovaginal probe with the needle held in the 
gun will be introduced into the vagina by the physician. The tip of the biopsy needle will be 
continuously visualized during the whole procedure for optimal sampling and ensure patient safety. 
The operator can use color/power Doppler US to increase the safety of the procedure by avoiding 
injuring large vessels when inserting the biopsy needle. In addition, Doppler US can help the operator 
identify a viable portion of the lesion without necrotic/cystic tissue, and thus minimize the risk of 
obtaining a sample inadequate for histological examination. When the needle is in a correct position 
and the penetration depth is checked, the gun will be activated by depressing the trigger button to fire 
the needle. After confirming successful tissue retrieval, the sample will be removed and placed into a 
labelled container with 10% buffered formalin. To ensure that the sample will be suitable for 
histological examination, we will require at least three passages of the needle into a neoplastic lesion 
[17, 18]. The collected tissue will be delivered to the Department of Pathology for examination and 
storage. At the end of the procedure, US should be continued to detect any signs of internal bleeding 
(e.g., “fountain sign”/turbulent flow) or other complications adjacent to the site of the biopsy. Mild 
internal bleeding from the biopsy site usually resolves spontaneously. If the patient suffers bleeding 
from the biopsy site in the vagina or in the vaginal wall, a hemostatic tamponade will be inserted inside 
the vagina. If an adverse event (AE) occurs, the patient will be hospitalized in the Oncogynecologic 
Department, depending on the severity of the AE. Data from TCB will be recorded using TCB 

13 
evaluation form, and details of AEs will be recorded using the AE evaluation form. AEs will be 
prospectively assessed according to the Clavien‒Dindo classification (Attachment 1) [19]. AEs will be 
reported separately according to whether they occurred during the procedure, within 24 hours after 
the procedure, or within 30 days after the procedure. Furthermore, the patients will be contacted by 
telephone within 72 hours to enquire about their subjective assessment of the procedure. 
 
4.1.2. Transabdominal/percutaneous TCB 
 
The patient will lie on a bed in the US room or operating theatre. Local anesthetic will be infiltrated 
into the abdominal wall. For patients without a known allergy, trimecaine will be applied as a local 
anesthetic, and injected immediately before performing the procedure, approximately 5 minutes 
before TCB. The needle will be inserted into an automatic biopsy gun. For the percutaneous 
approach, a 16G needle with a length of 20 cm will be used. After disinfecting the skin, the operator 
will puncture the skin with the needle in place in the biopsy gun held in one hand, with guidance with 
an abdominal probe placed in the operator’s other hand. The rest of procedure will be same as 
described in 4.1.1 Transvaginal TCB. If bleeding occurs, pressure will be applied to the site of 
bleeding. If pressure does not help, a stitch will be applied. AEs will be managed according to local 
recommendations. 
 
 
4.2. DESCRIPTION OF THE EXAMINATION METHODS 
4.2.1. Histopathological examination 
 
Standard histopathological examinations will be performed for all TCB samples. To guarantee the 
high quality of the results, only pathologists who are highly experienced with examining TCB samples 
will be responsible for the histopathological examinations and other examinations.  
 
4.2.2. Immunohistochemistry (IHC) 
 
IHC is a pathological method to detect specific tumor antigens. IHC uses monoclonal or polyclonal 
antibodies to determine the distribution of tumor antigens, which may be expressed de-novo or up-
regulated in a particular type of cancer [20]. IHC is often performed on tumors of unknown origin if the 
morphological results are inconclusive. Tumor cells may express specific intermediate filaments, such 
as keratin, desmin, vimentin, neurofilaments, and glial fibrillary acidic proteins [21]. By determining 
the expression of these and other proteins, IHC can precisely classify a tumor. Therefore, IHC is 
indicated by a pathologist in order to make an accurate diagnosis. Some gynecological tumors are 

14 
hormonally dependent, which means their growth is regulated by a hormone. Using IHC, we can 
determine the expression and distribution of hormonal receptors distribution, enabling more effective 
personalized treatment. IHC can also provide information about prognostic markers by identifying the 
enzymes, tumor-specific antigens, oncogenes, tumor suppressor genes, and tumor cell proliferation 
markers expressed by the tumor cells [20]. IHC can help us identify which patients are eligible for 
genetic screening (mismatch repair proteins – MLH1, PMS2, MSH2, MSH6 and Lynch syndrome 
screening) 
and 
to 
assess 
their 
potential 
eligibility 
for targeted 
therapies 
[22]. 
The 
gynecologist/oncologist may therefore indicate IHC to determine the patient’s prognosis and choose 
the appropriate treatment. Thus, a great deal of information can be obtain from IHC, as for NGS, and 
comparing the result can be valuable. 
 
4.2.3. Next-generation sequencing (NGS) 
 
NGS is massively parallel sequencing that enables us to identify previously unrecognized genomic 
alterations in many malignancies and enable targeted therapy. NGS can be targeted using DNA or 
RNA panels. Using NGS, we can identify germline and somatic mutations, molecular aberrations, and 
specific RNA fusions, as well as their associations with malignancies. NGS can also reveal the 
mechanism of resistance to specific treatments. However, the main limitation of of NGS is its cost. It 
is also questioned how often the result of NGS will have a clinical impact and will change the patient’s 
therapeutic strategy [22]. NGS-guided therapies include hormone therapies, pathway‐specific 
therapies, and immunotherapies, which are associated with longer progression‐free interval and 
longer overall survival. In gynecological tumors, the most commonly detected mutations are located 
in the genes TP53, MYC, KRAS and BRCA1/2. NGS examination and molecular classification can be 
performed at any time during the treatment course. Specific mutations can be detected in the tumor 
genome at initial diagnosis, or when can occur as acquired new resistance alterations after previous 
treatment lines. The results of NGS can optimize the therapeutic regimen and increase the opportunity 
for targeted therapy [23] [24]. For example, genomic profiling of endometrial cancers led to the 
reclassification of endometrial cancer into four groups that are distinct from the previous histological 
classification and are relevant to the consequent treatment of the patients [25]. 
NGS can also determine the tumor mutational burden (TMB), defined as the number of 
somatic mutations, that varies across malignancies. TMB can improve the predictive accuracy for 
immunotherapy outcomes, and has the potential to expand the number of candidates for treatment 
with immune checkpoint inhibitors [26]. 
We now have great expectations of liquid biopsy examination. Liquid biopsy is an essential 
non-invasive diagnostic test that evaluates circulating tumor cells and tumor DNA, as well as other 
blood markers that may be useful for guiding precision medicine. Although liquid biopsy is not a 

15 
routinely used diagnostic test, the potential applications in the diagnosis and prognosis in ovarian 
cancer are rapidly growing [27]. 
 
4.2.4. Immunologic examination 
 
It is accepted that the origin and development of cancer is enabled by the evasion of 
immunosurveillance due to the loss of antigenicity and/or immunogenicity, and by coordinating an 
immunosuppressive microenvironment [28] [29]. Specific oncological treatments, including 
chemotherapy, radiotherapy and immunotherapy, induce an immune response beyond the expected 
antitumor activity. This immune response can be evaluated by immunological testing. There are some 
immunological indicators that are relevant to the prognosis and prediction of a treatment response, 
including the density, functional state, and organization of tumor infiltrating lymphocytes (TIL) [30]. 
Specialized methods can be used to analyze the immune contexture of tumors, and to determine 
biomarkers that could permit the adaptation of individual treatment approaches, as well as monitoring 
the response to therapy [31]. Interesting information can also be obtained by comparing the T cell 
profiles of samples obtained at de novo diagnosis with those obtained upon recurrence after specific 
therapy and is therefore useful as part of the long-term follow-up. Immunologic examination of TCB 
will be done consequently after the standard pathologic tests (IHC and NGS) have been performed. 
 
 
 

16 
STUDY DESCRIPTION 
 
1. Referral to the Gynecologic Oncology Department 
Clinical examination 
Expert US 
2. Inclusion and exclusion criteria assessment 
Inclusion criteria  
1. Presence of a pelvic/abdominal lesion with suspicion of malignancy 
2. Age ≥18 years 
3. Eastern Cooperative Oncology Group performance status grade <3 
4. Not pregnant 
5. Signed informed consent form 
Exclusion criteria 
1. Considered by the investigator to be unsuitable for any treatment 
2. Early stage disease 
3. Age <18 years 
3. Informed consent signature 
Included: informed consent form signed 
Excluded: informed consent form not signed  
4. Registration on the TRUTH electronic database 
Clinical data form 
5. Index tests - US 
US evaluation form 
6. TCB 
TCB evaluation form 
AE evaluation form – including telephone call to identify AEs occurring within 72 hours after 
the procedure 
7. Histopathological report for TCB 
Pathology, NGS, immunohistochemistry, and immunology evaluation form 
8. Histopathological report from surgery 
Final pathology evaluation form 
AE: adverse event; NGS: next-generation sequencing TCB: tru-cut biopsy; US: ultrasound 
 
 

17 
Patients 
undergoing TCB 
Patients with an 
adequate sample 
Patients enrolled 
in the study 
Patients 
undergoing surgery 
Figure 1 Flow chart describing the design and patient flow in the TRUTH trial 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patients not suitable 
for TCB 
Patients in whom an 
adequate sample was 
not obtained 
Patients not undergoing 
surgery 
FEASIBILITY 
ADEQUACY 
ACCURACY 
SAFETY 

18 
5. STATISTICAL ANALYSIS  
 
The power analysis was computed for adequacy/feasibility of immunological examination based on 
published data and a pilot study for samples obtained by TCB.  
The sample size analysis was calculated for adequacy of samples obtained by TCB for 
histopathological diagnosis. The calculation was computed under the assumption of sample 
adequacy between 50-90%. The evaluated parameter was the width of 95% confidence interval for 
the proportion of adequate sample (Table 1-2). 
With the expected adequacy of the samples in the range of 70-90%, we plan to enroll at 
minimum 250 subjects, in order to achieve confidence interval of ≤10%. 
The analysis was computed using PASS 16 Power Analysis and Sample Size Software (2018) 
NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/pass, by Institute of Biostatistics and Analyses 
(Faculty of Medicine, Masaryk University, Brno). [32, 33] 
 
Standard descriptive statistics will be applied for the exploratory analysis, including absolute 
and relative frequencies for categorical variables and medians with 5th–95th percentiles and/or means 
with standard deviations or confidence intervals (95%) of point estimates. The statistical significance 
of differences in categorical variables will be assessed using the maximum likelihood chi-square test. 
The accuracy, adequacy, and safety will be assessed by performing receiver operator characteristic 
(ROC) curve analysis, after selecting appropriate predictors, and with a standard set of descriptive 
statistics alongside confidence intervals related to the individual predictors (area under the curve, 
sensitivity, specificity, positive and negative predictive value, overall accuracy). 
Statistical analyses will be performed using SPSS 24.0.0.0 (IBM Corporation, 2015) statistical 
software. 
 
6. STUDY TIMELINE AND SAMPLE SIZE 
 
At Gynecologic Oncology Centre of the General University Hospital in Prague, approximately 100 
women undergo TCB per year. According to the power analysis, the required study sample size is 
250 cases. Therefore, the required number of the patients undergoing TCB is expected to be achieved 
in 2-3 years. All participants will be followed-up for 30 days after TCB to monitor any AEs. The total 
duration of the project is 3 years, depending on the main endpoint and power analysis.  
 
 
 
 

19 
7. EXPECTED OUTCOMES 
 
This prospective study is to our knowledge the first study registered on www.clinicaltrials.gov 
designed to prospectively evaluate the quality of samples obtained by TCB for comprehensive 
classification of pelvic and abdominal malignant tumors by IHC, NGS, and immunological evaluation, 
and allowing evaluation based on location, size, and other factors. Our study is also expected to 
confirm the known feasibility and safety of TCB, and demonstrate its high adequacy and accuracy. 
The findings should also provide an update to the promising results of a previous retrospective 
analysis of TCB in a larger study population.  
 
 
8. ETHICAL COMMITTEE APPROVAL  
 
Before the start of the study, this protocol and other related documents must be submitted for review 
to the Ethical Committee for authorization. The study will not commence until such approvals have 
been obtained and until other relevant essential documents, such as the signed contract agreements 
and evidence of adequate study financing, are prepared. 
The study must be performed in accordance with the protocol, current ICH and ICH-GCP 
guidelines, and applicable regulatory and country-specific requirements. ICH guidelines are an 
international ethical and scientific quality standard for designing, conducting, recording, and reporting 
studies that involve the participation of human participants. Compliance with this standard provides 
public assurance that the rights, safety, and well-being of participants are protected, consistent with 
the principles that originated in the most recent version of the Declaration of Helsinki, and that the 
study data are credible, reliable and reproducible. 
The study will be conducted in compliance with the requirements of the EU General Data 
Protection Regulation 2016/679 (GDPR). Any personal data will always be treated as confidential 
including during collection, handling and use, or processing, and the personal data (including in any 
electronic format) shall always be stored securely, with all technical and organizational security 
measures necessary for compliance with EU and national data protection legislation (whichever is 
more stringent). The sponsor shall take appropriate measures to ensure the security of all personal 
data and guard against unauthorized access thereto or disclosure thereof or loss or destruction while 
in its custody. 
 
 
 
 

20 
9. DATA HANDLING AND MONITORING 
 
Data collection, handling, processing, and transfer for the purpose of the study will be performed in 
compliance with applicable regulations, guidelines for clinical studies and internal procedures. It 
remains the responsibility of the investigator to check that all data are entered into the electronic Case 
Report Form (eCRF) in accordance with the instructions provided and that the forms are filled out 
accurately, completely, and in a timely manner. 
The study will use the REDCap (Research Electronic Data Capture, www.project-redcap.org) 
electronic data capture tool. It is a secure, web-based application with controlled access designed to 
support data capture for research studies, hosted at MD Anderson. Access will be restricted to 
authorized users only. 
The study will be monitored by the study coordinator and the main investigator, who are 
responsible for checking the accuracy, completeness, and plausibility of all data, as well as 
compliance the study protocol and GCP requirements. 
 
 
10.   FUNDING AND SUPPORT  
 
The TRUTH study is a non-commercial trial that is not receiving any support from the industry. The 
participating site and patients will not receive any financial compensation for participation in the study. 
All expenses related to the trial (administration, statistics, electronic data capture system, monitoring) 
will be covered by a research grant. 
Patients enrolled in the TRUTH trial will be insured according to the laws and regulations of 
our country TCB and the new examination methods are considered routine procedures as part of the 
standard workup, including disease recurrence/progression, for evaluation of tumour characteristics 
according to international guidelines. Therefore, procedures are reimbursed as part of standard 
clinical care. No separate insurance is requested for the project. 
 
 
11.  PUBLICATION PLAN 
 
All publications will be coordinated by the main investigator. Authorship will be determined in 
accordance with the requirements published by the International Committee of Medical Journal 
Editors and the requirements of the respective medical journal. 
 
 

21 
12.  
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