Ignite Creation Date:
2024-05-06 @ 3:55 PM
Last Modification Date:
2024-10-26 @ 1:59 PM
Study NCT ID:
NCT04800497
Status:
RECRUITING
Last Update Posted:
2024-01-17
First Post:
2021-02-22
Brief Title:
The Role Of Circulating Tumor Cells As Markers Of Advanced Disease And Prognosis In HCC
Sponsor:
University of Milano Bicocca
Organization:
University of Milano Bicocca
Study Overview
Official Title:
Finding The Seeds Of Recurrence The Role Of The Liquid Biopsy To Detect Circulating Tumor Cells As Markers Of Advanced Disease And Prognosis In HCC
Status:
RECRUITING
Status Verified Date:
2024-01
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Hepatocellular carcinoma HCC recurs in up to 60 of patients who undergo resection Circulating tumor cells CTC have been advocated as promotors of the recurrence However their role as prognostic markers in the surgical setting is unclear The aim of the present study has been to assess the association between CTC from peripheral blood samples and the risk of recurrence after surgery
Patients with a first diagnosis of HCC no previous treatment for this condition no other oncological history and BCLC stage 0-A-B will be enrolled in 2 centers Patients will undergo to serial liquid biopsies ie a 15ml peripheral blood sample on each time point at day 0-30-90-180-365 after surgery After isolation of peripheral blood mononucleate cells CTC will be detected by FACSymphony and subsequently the following markers will be identified EpCAM N-cadherin N-cad and CD90 Epithelial-mesenchymal transition EMT will be analyzed by an index estimated as the ratio between the number of EpCAMN-cad- and EpCAMN-cad cells EMT Index Patients will be divided according to the recurrence status
Patients with a first diagnosis of HCC no previous treatment for this condition no other oncological history and BCLC stage 0-A-B will be enrolled in 2 centers Patients will undergo to serial liquid biopsies ie a 15ml peripheral blood sample on each time point at day 0-30-90-180-365 after surgery After isolation of peripheral blood mononucleate cells CTC will be detected by FACSymphony and subsequently the following markers will be identified EpCAM N-cadherin N-cad and CD90 Epithelial-mesenchymal transition EMT will be analyzed by an index estimated as the ratio between the number of EpCAMN-cad- and EpCAMN-cad cells EMT Index Patients will be divided according to the recurrence status
Detailed Description:
Liquid biopsy may allow a better stratification of patients according to their estimated prognosis As well in other tumors eg breast cancer molecular characterization is permitting not only to better understand the pathological pathways which lead tumorigenesis and progression but also to drive specific treatments tailored on the specificity of the patient disease Liquid biopsy is very easy and cheap to perform since it consist in a simple venous blood sample which will be investigated by cells sorter to identify specific populations of cells
In hepatocellular carcinoma is common to assist on an elevated recurrence rate patients who have been treated with a curative intent in 70 of cases relapse In these cases patients often had an underlying inflammatory liver disease as cirrhosis which may drive the relapse as an oncogenetic factor Cirrhosis is a well-know risk factor for developing hepatocellular carcinoma in general Because of this in clinical practice is almost difficult to distinguish if the new nodule we have diagnosed should be considered as a real recurrence of the first treated tumor or a de-novo occurrence linked to the unchanged underlying tumorigenesis boost which may be effectively cured only after liver transplantation Several retrospective studies already demonstrated the advantage in terms of disease-free-survival and overall-survival of a de-novo occurrence in respect to a real metastatic presentation However no one has reached any specific clinical or morphological markers to distinguish between these two conditions Characterization of the nature of the recurrent nodule has been obtained only by genetic and molecular evaluation of the tumors heterogeneity Nevertheless this characterization nowadays is possible only with research intent and on histological specimen which could be provided only after an eventual decision of redo-surgery to treat the considered recurrence Indication on how to treat HCC recurrence after surgery still lacks Our hypothesis is founded on the seed and oil theory which is basically founded on the evidence of the presence of circulating tumor cells CTCs in the blood stream These cells may be spread from the original tumors as an hallmark of advance cancer which has already developed the invasiveness characteristic It is our opinion that tumors in an early stage do not release or not in the same rate or quality CTCs in the bloodstream as advanced tumor does We considered an advanced tumor the one which may has already microvascular invasion andor satellitosis which may be the histological signs of an advanced and already systemic stage Patients with CTCs identified at the liquid biopsy may be associated with a worsen prognosis accounting for an increased rate of early recurrence and overall recurrence even in case of transplantation In addition the seed and oil theory associated with the rehoming theory may justified from a bio-oncological point of view the cases of recurrence in the transplant setting when the whole organ and the relative oncogenetic risk factors is treated by substitution Finally from a pathophysiological point of view we want to demonstrate that recurrent intra-hepatic nodules IM are due to circulating tumour cellsseeding permitting a better understanding of carcinogenesis in those tumours
Primary objective to evaluate the association between variation in CTCs - determined in blood samples - and overall survival and disease-free-survival in HCC patients before and after surgery either resection and transplant
Secondary objective to evaluate if the positive identification of CTCs in blood samples is associated with the intra-hepatic metastatic pattern of recurrence in either resected patients and transplanted one
Tertiary objective to evaluate and to monitor the role of different clinical biochemical radiological and histopathological variables in determining the recurrence after surgery and their eventual relationship with the presence and rate of CTCs Patients enrollment will be assessed by the surgical divisions of this study usually during the outpatient clinic visit before the surgical operation In that moment patients will be candidate for this study and if they accept to participate researchers will acquire the written consent Enrolled patients will be codified by an alpha-numeric number to preserve the identity Researchers who will be employed in the laboratory where the CTCs will be analyzed will not be able to trace the owner of the sample and they will not have the access to the clinical data blind analysis
Before surgery for each patient liquid biopsy from peripheral blood will be collected in the same time of routine blood analysis Samples will be used to analyze CTCs rate and type by FACSARIA III BD bioscience During surgery a fresh slice of the tumor and near health liver will be collected by an expert pathologist who will use sections for the usual histological evaluation as provided by the best clinical practice and international guidelines The remaining sections will be maintained on ice during shipment to the laboratory unit Researchers will perform alive hepatic cells isolation and storage under sterile conditions Then hepatic cells will be analyzed with FACSARIA III and used for further in-vitro experiments
Liquid biopsy will be performed again at 30th post-operative day during the first outpatient visit and then every six months in accordance and in parallel with the local follow-up protocols Liquid biopsy performed during follow-up will be managed as the pre-operative one during the normal tests provided for clinical purpose Follow-up will be closed at 30 months
All operations will be performed by surgeons with large experience in liver surgery The resection technique will be at surgeon preference according to local protocol good clinical practise and national guidelines All surgeons adopted the same criteria to select the surgical strategy baseline variables of liver function tumour location and diameter and number of nodules Restaging Ultrasonography US will be performed intra-operatively for each patient After tumor removal a pathologist involved in this study will sample the tumor to obtain a fresh-frozen section of the tumor with the related healthy liver tissue to store it for further analysis This sample should be performed in accordance with international guidelines and local ethical agreement and it must always guarantee the correct material to perform the histological diagnosis If the tissue removed from the patient enrolled in this study does not allow to perform the sample for the study without affecting the optimal histological diagnosis the patient will be excluded from the study and no sampling will be executed
For all patients 20 mL fasting blood sample will be collected in a sterile vacuum tube The collection will be done in the same time of routine preoperative blood samples or planned follow-up controls since no others invasive procedure should be provided to patients since their enrollment in the study The blood sample will be stored at 4C and handled within 6 hours Then 5 mL will be centrifuged at 3000 rpm for 10 minutes at 4C the plasma phase will be collected and aliquoted in 15 mL sterile tubes and stored at -80C until use or shipment Then we will isolate peripheral blood mononucleate cells PBMC through Ficoll gradient from the remaining 15 mL After isolation live PBMC will be frozen in standard media 20 Fetal Bovin Serum FBS 10 Dymethil Sulfoxide DMSO and stored in liquid nitrogen until use
For all patient liver tissue sample will be collected and tumor T portion will be separated from peritumor PT portion From T and PT tissue will be isolated consequentially Human Hepatocytes HC Kupffer cells KC Liver Sinusoidal Endothelial Cells LSEC and Human Stellate Cells HSC under sterile conditions Briefly liver tissue will be placed in a Petri dish and cut with sterile scalpel Then cut liver will be incubated in a 50 mL tube with collagenase P 1mgmL at 37C for 45 minutes After incubation collagenase P will be inactivated adding 20 FBS Hanks Balance Salt Solution HBSS and cell suspension will be centrifuged 50g 5 minutes at 4C The supernatant containing liver non-parenchymal cells NPC will be collected in a new 50 mL tube and pellet will be washed with Phosphate Buffer Solution PBS HC and NPC will be frozen in standard media 20 FBS 10 DMSO and stored in liquid nitrogen until use
A fragment of T and PT will be stored in RNAlater solution at -80C For CTC identification PBMC will be thawed resuspended in standard media and sorted with FACSARIA III BD bioscience through EpCAM CD47 CD133 The percentage of single positive cells EpCAM CD47 and CD133 and triple positive cells EpCAMCD47CD133 will be evaluated and the total CTC number will be calculated CTC will be tested for their replicative and invasive capacity Using cell proliferation and invasion assay we will evaluate CTC doubling time time needed for the cells to double in number growth rate grlnNtN0t Nt the number of cells at time t N0 the number of cells at time 0 t time and invasion rate
After CTC characterization we will analyse HC and NPC using the positive markers previously described in order to evaluate the exact CTC hepatic derivation
The biological material will be shipped by expert private transport and stored to the laboratory in Università degli Studi del Piemonte Orientale
The recurrence patterns will be evaluated as follow Local recurrence will be defined as a recurrence on the same liver segment in case of Parenchyma Sparing Resection or on the surgical hedge of the nearest segments in case of Anatomic Resection Thus it will be evaluated the number of recurrent nodules single versus multiple the location of recurrence intrahepatic versus extrahepatic or both the size of the main recurrent nodule and the liver segment All this evaluations on recurrence will be made by CT scan or MRI scan during follow-up as the local follow-up protocols provide
All the patients will be followed using a local protocol including measurement of serum α-FP total bilirubin GGT ALP Albumine INR blood count electrolyte count ultrasound contrast Computed Tomography CT or magnetic Resonance Imaging MRI and office visits Patients usually are checked every 3 months during the first postoperative year and every 6 months thereafter When the recurrence occurred the cases are discussed in a multidisciplinary setting involving radiologist hepatologist surgeon interventional radiologist radiotherapist and oncologist Overall-Survival OS is defined as time interval in months from surgery to patient death Data will be censored in case of loosed patient at the follow-up screening Disease-Free Survival DFS is defined as time interval in months from surgery to recurrence or death In case of no recurrence data will be censored at the date of the last available follow-up Tumor-Specific-Survival TSS is defined as the time interval in months between surgery and tumor-related death
In hepatocellular carcinoma is common to assist on an elevated recurrence rate patients who have been treated with a curative intent in 70 of cases relapse In these cases patients often had an underlying inflammatory liver disease as cirrhosis which may drive the relapse as an oncogenetic factor Cirrhosis is a well-know risk factor for developing hepatocellular carcinoma in general Because of this in clinical practice is almost difficult to distinguish if the new nodule we have diagnosed should be considered as a real recurrence of the first treated tumor or a de-novo occurrence linked to the unchanged underlying tumorigenesis boost which may be effectively cured only after liver transplantation Several retrospective studies already demonstrated the advantage in terms of disease-free-survival and overall-survival of a de-novo occurrence in respect to a real metastatic presentation However no one has reached any specific clinical or morphological markers to distinguish between these two conditions Characterization of the nature of the recurrent nodule has been obtained only by genetic and molecular evaluation of the tumors heterogeneity Nevertheless this characterization nowadays is possible only with research intent and on histological specimen which could be provided only after an eventual decision of redo-surgery to treat the considered recurrence Indication on how to treat HCC recurrence after surgery still lacks Our hypothesis is founded on the seed and oil theory which is basically founded on the evidence of the presence of circulating tumor cells CTCs in the blood stream These cells may be spread from the original tumors as an hallmark of advance cancer which has already developed the invasiveness characteristic It is our opinion that tumors in an early stage do not release or not in the same rate or quality CTCs in the bloodstream as advanced tumor does We considered an advanced tumor the one which may has already microvascular invasion andor satellitosis which may be the histological signs of an advanced and already systemic stage Patients with CTCs identified at the liquid biopsy may be associated with a worsen prognosis accounting for an increased rate of early recurrence and overall recurrence even in case of transplantation In addition the seed and oil theory associated with the rehoming theory may justified from a bio-oncological point of view the cases of recurrence in the transplant setting when the whole organ and the relative oncogenetic risk factors is treated by substitution Finally from a pathophysiological point of view we want to demonstrate that recurrent intra-hepatic nodules IM are due to circulating tumour cellsseeding permitting a better understanding of carcinogenesis in those tumours
Primary objective to evaluate the association between variation in CTCs - determined in blood samples - and overall survival and disease-free-survival in HCC patients before and after surgery either resection and transplant
Secondary objective to evaluate if the positive identification of CTCs in blood samples is associated with the intra-hepatic metastatic pattern of recurrence in either resected patients and transplanted one
Tertiary objective to evaluate and to monitor the role of different clinical biochemical radiological and histopathological variables in determining the recurrence after surgery and their eventual relationship with the presence and rate of CTCs Patients enrollment will be assessed by the surgical divisions of this study usually during the outpatient clinic visit before the surgical operation In that moment patients will be candidate for this study and if they accept to participate researchers will acquire the written consent Enrolled patients will be codified by an alpha-numeric number to preserve the identity Researchers who will be employed in the laboratory where the CTCs will be analyzed will not be able to trace the owner of the sample and they will not have the access to the clinical data blind analysis
Before surgery for each patient liquid biopsy from peripheral blood will be collected in the same time of routine blood analysis Samples will be used to analyze CTCs rate and type by FACSARIA III BD bioscience During surgery a fresh slice of the tumor and near health liver will be collected by an expert pathologist who will use sections for the usual histological evaluation as provided by the best clinical practice and international guidelines The remaining sections will be maintained on ice during shipment to the laboratory unit Researchers will perform alive hepatic cells isolation and storage under sterile conditions Then hepatic cells will be analyzed with FACSARIA III and used for further in-vitro experiments
Liquid biopsy will be performed again at 30th post-operative day during the first outpatient visit and then every six months in accordance and in parallel with the local follow-up protocols Liquid biopsy performed during follow-up will be managed as the pre-operative one during the normal tests provided for clinical purpose Follow-up will be closed at 30 months
All operations will be performed by surgeons with large experience in liver surgery The resection technique will be at surgeon preference according to local protocol good clinical practise and national guidelines All surgeons adopted the same criteria to select the surgical strategy baseline variables of liver function tumour location and diameter and number of nodules Restaging Ultrasonography US will be performed intra-operatively for each patient After tumor removal a pathologist involved in this study will sample the tumor to obtain a fresh-frozen section of the tumor with the related healthy liver tissue to store it for further analysis This sample should be performed in accordance with international guidelines and local ethical agreement and it must always guarantee the correct material to perform the histological diagnosis If the tissue removed from the patient enrolled in this study does not allow to perform the sample for the study without affecting the optimal histological diagnosis the patient will be excluded from the study and no sampling will be executed
For all patients 20 mL fasting blood sample will be collected in a sterile vacuum tube The collection will be done in the same time of routine preoperative blood samples or planned follow-up controls since no others invasive procedure should be provided to patients since their enrollment in the study The blood sample will be stored at 4C and handled within 6 hours Then 5 mL will be centrifuged at 3000 rpm for 10 minutes at 4C the plasma phase will be collected and aliquoted in 15 mL sterile tubes and stored at -80C until use or shipment Then we will isolate peripheral blood mononucleate cells PBMC through Ficoll gradient from the remaining 15 mL After isolation live PBMC will be frozen in standard media 20 Fetal Bovin Serum FBS 10 Dymethil Sulfoxide DMSO and stored in liquid nitrogen until use
For all patient liver tissue sample will be collected and tumor T portion will be separated from peritumor PT portion From T and PT tissue will be isolated consequentially Human Hepatocytes HC Kupffer cells KC Liver Sinusoidal Endothelial Cells LSEC and Human Stellate Cells HSC under sterile conditions Briefly liver tissue will be placed in a Petri dish and cut with sterile scalpel Then cut liver will be incubated in a 50 mL tube with collagenase P 1mgmL at 37C for 45 minutes After incubation collagenase P will be inactivated adding 20 FBS Hanks Balance Salt Solution HBSS and cell suspension will be centrifuged 50g 5 minutes at 4C The supernatant containing liver non-parenchymal cells NPC will be collected in a new 50 mL tube and pellet will be washed with Phosphate Buffer Solution PBS HC and NPC will be frozen in standard media 20 FBS 10 DMSO and stored in liquid nitrogen until use
A fragment of T and PT will be stored in RNAlater solution at -80C For CTC identification PBMC will be thawed resuspended in standard media and sorted with FACSARIA III BD bioscience through EpCAM CD47 CD133 The percentage of single positive cells EpCAM CD47 and CD133 and triple positive cells EpCAMCD47CD133 will be evaluated and the total CTC number will be calculated CTC will be tested for their replicative and invasive capacity Using cell proliferation and invasion assay we will evaluate CTC doubling time time needed for the cells to double in number growth rate grlnNtN0t Nt the number of cells at time t N0 the number of cells at time 0 t time and invasion rate
After CTC characterization we will analyse HC and NPC using the positive markers previously described in order to evaluate the exact CTC hepatic derivation
The biological material will be shipped by expert private transport and stored to the laboratory in Università degli Studi del Piemonte Orientale
The recurrence patterns will be evaluated as follow Local recurrence will be defined as a recurrence on the same liver segment in case of Parenchyma Sparing Resection or on the surgical hedge of the nearest segments in case of Anatomic Resection Thus it will be evaluated the number of recurrent nodules single versus multiple the location of recurrence intrahepatic versus extrahepatic or both the size of the main recurrent nodule and the liver segment All this evaluations on recurrence will be made by CT scan or MRI scan during follow-up as the local follow-up protocols provide
All the patients will be followed using a local protocol including measurement of serum α-FP total bilirubin GGT ALP Albumine INR blood count electrolyte count ultrasound contrast Computed Tomography CT or magnetic Resonance Imaging MRI and office visits Patients usually are checked every 3 months during the first postoperative year and every 6 months thereafter When the recurrence occurred the cases are discussed in a multidisciplinary setting involving radiologist hepatologist surgeon interventional radiologist radiotherapist and oncologist Overall-Survival OS is defined as time interval in months from surgery to patient death Data will be censored in case of loosed patient at the follow-up screening Disease-Free Survival DFS is defined as time interval in months from surgery to recurrence or death In case of no recurrence data will be censored at the date of the last available follow-up Tumor-Specific-Survival TSS is defined as the time interval in months between surgery and tumor-related death
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None