Ignite Creation Date:
2024-05-06 @ 3:51 PM
Last Modification Date:
2024-10-26 @ 1:58 PM
Study NCT ID:
NCT04775862
Status:
UNKNOWN
Last Update Posted:
2021-03-01
First Post:
2021-02-21
Brief Title:
A Prospective Study Utilizing Circulating Cell Free DNA cfDNA Use in the Detection of RAS Mutations in Patients With Advanced Colorectal Cancer
Sponsor:
National Guard Health Affairs
Organization:
National Guard Health Affairs
Study Overview
Official Title:
A Prospective Phase II Study Utilizing Circulating Cell Free DNA cfDNA Use in the Detection of RAS Mutations in Patients With Advanced Colorectal Cancer
Status:
UNKNOWN
Status Verified Date:
2021-02
Last Known Status:
RECRUITING
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:
Colorectal cancer remains the commonest cancer among men and third commonest among women in Saudi Arabia Presentation with metastatic disease occurs in almost one third of patients with 5-year survival decreasing significantly from 90 in stage 1 to 14 once the disease is metastatic There is enthusiasm in the potential for liquid biopsies to provide easily accessible genetic biomarkers for mutational cancer characterization Epidermal growth factor receptor EGFR monoclonal antibodies are widely used in the treatment of advanced colorectal cancer that do not harbor RAS mutations RAS wild type Hence genotyping of oncogenic RAS mutations is essential prior to the initiation of systemic therapy for such patients as the presence of these mutations predict resistance to EGFR targeted antibodies such as Cetuximab and Panitumumab Detection of such mutations has been done on tissue biopsies with the disadvantage of this being an invasive procedure and data suggesting that such testing may not be reflective of the true mutational burden of the disease since a single fragment of tissue may be inadequate to reflect the intratumoral heterogeneity There is increasing evidence suggesting that liquid biopsies or blood based mutational profiling can provide a more comprehensive molecular profile of the disease and carries the advantage of being minimally invasive Serial liquid biopsies can act as a tool to identify spatial and temporal heterogeneity predicting response or resistance to targeted agents and can shed light into the emergence or disappearance of specific mutations that may potentially be targeted with newer anti cancer agents
Circulating cell free DNA cfDNA consists of small nucleic acid fragments liberated from cells by rupture necrosis or apoptosis and is now increasingly being used to detect RAS and other mutations in patients with advanced colorectal cancers KRAS has remained an undruggable target for decades until the most recent evidence that showed a new anticancer drug that targets KRAS G12C mutation
The investigators aim to perform cfDNA testing on patients with advanced colorectal cancers who have no RAS mutations and hence start on EGFR inhibitors as baseline compare the results with mutational analysis on fresh tumor tissue and perform cfDNA at first progression to determine what mutations have emerged and specifically look for KRAS G12C mutation which can be targeted with a new novel anti cancer drug These patients will be collected over a 12 month period with the aim of performing this on at least 100 patients and followed from diagnosis with baseline cfDNA and until progression on EGFR inhibitors where another cfDNA sample will be taken A detailed proposal delineating this process will follow once accepted
This project is unique as it examines mechanisms of resistance to anti-EGFR inhibitors in our patients with advanced colorectal cancers determines the prevalence of a specific mutation using liquid biopsies and examining cfDNA use and may have therapeutic implications in facilitating obtaining KRAS G12C inhibitors for such patients
Circulating cell free DNA cfDNA consists of small nucleic acid fragments liberated from cells by rupture necrosis or apoptosis and is now increasingly being used to detect RAS and other mutations in patients with advanced colorectal cancers KRAS has remained an undruggable target for decades until the most recent evidence that showed a new anticancer drug that targets KRAS G12C mutation
The investigators aim to perform cfDNA testing on patients with advanced colorectal cancers who have no RAS mutations and hence start on EGFR inhibitors as baseline compare the results with mutational analysis on fresh tumor tissue and perform cfDNA at first progression to determine what mutations have emerged and specifically look for KRAS G12C mutation which can be targeted with a new novel anti cancer drug These patients will be collected over a 12 month period with the aim of performing this on at least 100 patients and followed from diagnosis with baseline cfDNA and until progression on EGFR inhibitors where another cfDNA sample will be taken A detailed proposal delineating this process will follow once accepted
This project is unique as it examines mechanisms of resistance to anti-EGFR inhibitors in our patients with advanced colorectal cancers determines the prevalence of a specific mutation using liquid biopsies and examining cfDNA use and may have therapeutic implications in facilitating obtaining KRAS G12C inhibitors for such patients
Detailed Description:
Colorectal cancer remains the commonest cancer among men and third commonest among women in Saudi Arabia Presentation with metastatic disease occurs in almost one third of patients with 5-year survival decreasing significantly from 90 in stage 1 to 14 once the disease is metastatic There is enthusiasm in the potential for liquid biopsies to provide easily accessible genetic biomarkers for mutational cancer characterization Epidermal growth factor receptor EGFR monoclonal antibodies are widely used in the treatment of advanced colorectal cancer that do not harbor RAS mutations RAS wild type Hence genotyping of oncogenic RAS mutations is essential to be done prior to initiation of systemic therapy for such patients as the presence of these mutations predict resistance to EGFR targeted antibodies such as cetuximab and panitumumab Treatment of metastatic CRC has become more complex and precision medicine approaches have evolved in recent years with the discovery of new oncogenic potentially targetable pathways The prognosis of metastatic colorectal cancer has improved from 6 months with best supportive care to more than 2 years with multi-agent chemo and targeted therapy including anti EGFR antibodies Targeting other singling pathways in CRC such as adding vascular endothelial growth factor inhibitors has benefitted patients as well It is estimated that 55 of patients with metastatic colorectal cancer mCRC will have oncogenic mutations in KRAS and NRAS Detection of such mutations has been done on tissue biopsies with the disadvantage of this being an invasive procedure and data suggesting that such testing may not be reflective of the true mutational burden of the disease since a single fragment of tissue may be inadequate to reflect the intratumoral heterogeneity There is increasing evidence suggesting that liquid biopsies or blood based mutational profiling can provide a more comprehensive molecular profile of the disease and carries the advantage of being minimally invasive Serial liquid biopsies can act as a tool to identify spatial and temporal heterogeneity predicting response or resistance to targeted agents and can shed light into the emergence or disappearance of specific mutations that may potentially be targeted with newer anti cancer agents To account for this molecular heterogeneity the genomic profiles of metastatic colorectal cancer patients should be examined at different time points during the course of therapy using liquid biopsy
There have been small studies that examined mechanisms of resistance to anti EGFR monoclonal antibodies in mCRC using liquid biopsy A study of 37 mCRC patients who were treated with cetuximab found that 40 of them developed RAS mutations at progression 10 Another study with limited number of participants examined patients with mCRC treated with panitumumab and found that 9 out of 24 patients 38 developed KRAS mutations on treatment as a mechanism of acquired resistance to anti EGFR therapy Furthermore fewer studies with limited number of patients used liquid biopsy as a biomarker when re-challenging mCRC patients with EGFR monoclonal antibodies The majority of these studies were retrospective However one was the first prospective trial and had a similar protocol to our study It included 28 patients and reported that 52 of these patients were RAS wildtype at re-challenge with cetuximab - when these patients were exposed to and progressed on cetuximab in the first line setting This study showed that re-challenge with cetuximab significantly improved progression free survival when RAS was found to be wild type on circulating tumor DNA12 One of the limitations of this study was that a single liquid biopsy sample was done prior to re-challenge with cetuximab and hence does not display the predicted switch of the RAS target which the investigators plan to study in our trial Furthermore a more recent study protocol has been published at BMC Cancer where the investigators plan to study 120 patients and perform liquid biopsy analysis every 3 months while patients are on first line cetuximab This is to study the evolution of the RAS target and to correlate this with disease response as well as help guide therapy with EGFR inhibitors in mCRC patients However based on limited data current guidelines have not yet adopted testing using liquid biopsy and using this strategy to decide on re challenge of anti EGFR therapy in 3rd line setting or not which is the question investigators would like to answer in this study
Circulating cell free DNA cfDNA consists of small nucleic acid fragments liberated from cells by rupture necrosis or apoptosis originating from normal and deceased cells and is now increasingly being used to detect RAS and other mutations in patients with advanced colorectal cancers There is new evidence that G12C RAS mutation can be targeted with a novel anti cancer agent
the investigators aim to perform cfDNA testing on patients with advanced colorectal cancers who have no RAS mutations ie wild type and hence start on EGFR inhibitors - which is standard of care treatment pre third line therapy This will help the treating physician decide whether to give these patients with RAS wt status an anti-EGFR monoclonal antibody or standard third line therapy Regorafenib or TAS-102 These patients will be collected over an 18 month period The cfDNA test at second progression ie prior to third line systemic therapy will determine whether the subset of patients who may have developed RAS mutations after progression to first line therapy or other mutations as a mechanism of resistance with anti - EGFR monoclonal antibodies have switched their RAS status and became wild type This will support the re-challenge of EGFR inhibitors in the third line setting and has the potential of changing the colorectal cancer treatment guidelines Upon this the principle investigator will decide whether to re-challenge with anti EGFR inhibitor The investigators aim to study 60 patients in total and have 30 patients at least in the rechallenge with anti EGFR mAb group
Materials and Methods
Patients will have their standard of care SOC biopsy of tumor metastatic site to confirm diagnosis and determine RAS status Once RAS wild type and primary disease is left sided these patients will receive standard chemotherapy choices of FOLFOX FLOFIRI CapeOX XELIRI with an anti EGFR mAb cetuximab or panitumumab Upon progression of disease second line systemic chemotherapy - anti VEGF antibody will be given as per SOC Upon second progression patients will be enrolled into the study as per inclusion criteria and consent and a cfDNA blood test will be drawn and RAS status will be examined If RAS is wildtype then the investigator will decide whether to re-challenge with an anti EGFR antibody see study schema - figure 1 or give SOC third line chemotherapy Regorafenib or TAS-102
Disease assessments will be done every 8 - 12 weeks as per SOC using CT scans andor MRI and will be reported as per RECIST criteria v11
Methods for cfDNA Next Generation Sequencing NGS from CRC patients cfDNA extraction Blood samples will be collected in K2EDTA tubes BD Vacutainer Blood Collection Tubes Becton Dickinson Franklin Lakes USA and sent to the Translational Pathology Laboratory The plasma fraction will be separated from the blood cells by two consecutive rounds of centrifugation for 30 min at room temperature at 1600 g The collected plasma was aliquoted and stored at -80 C until use cfDNA is extracted from plasma volumes ranging from 04 to 55 ml using the MagMax Cell-Free Total Nucleic Acid Isolation Kit Thermo Fisher Scientific Waltham USA according to the manufacturers instructions The cfDNA quantity was assessed with the dsDNA HS assay kit by the Qubit 20 Fluorometer Thermo Fisher Scientific cfDNA quality was assessed with the Agilent Tap Station System Agilent Technologies Santa Clara USA Only cfDNA samples with a clear fragment size peak between 140-200 bp will be considered for analysis
NGS library preparation NGS libraries will prepared from 10 ng of cfDNA following the Oncomine Pan-Cancer Cell-Free Assay Thermo Fisher Scientific Our general library preparation protocol is based on a two-cycle multiplex touch-down PCR reaction with a temperature range from 64 C to 58 C which allowed to amplify target regions and introduce unique molecular identifiers The resulting tagged amplicons of around 100-140 bp length are then cleaned up using Agencourt AMPure XP Beckman Coulter Brea USA at a bead to sample ratio of 15 and purified products are eluted in 24 μl low TE buffer A second round of PCR 18 cycles will be performed in a total volume of 50 μl to amplify the purified amplicons and introduce Ion Torrent Tag-Sequencing adapters containing sample-specific barcodes The resulting library of target DNA fragments will be purified by performing a two-step cleanup using Agencourt AMPure XP Beckman Coulter at a bead to sample ratio of 115 and 10 respectively The purified libraries re then diluted 11000 and quantified by qPCR using the Ion Universal Quantitation Kit Thermo Fisher Scientific The quantified stock libraries are then diluted to 100 pM for downstream template preparation
Sequencing NGS libraries will be sequenced on an Ion S5 instrument Thermo Fisher Scientific using semiconductor sequencing technology Briefly sequencing runs are planned on the Torrent Suite Software v510 libraries are pooled and loaded on an Ion 540 chip using the Ion Chef instrument Thermo Fisher Scientific The loaded chip is then sequenced using 500 flows Raw data are processed automatically on the Torrent Server and aligned to the reference hg19 genome QC will be performed manually for each sample based on the following metrics number of reads per sample15000000 for Oncomine Pan-Cancer Cell-Free Assay libraries aries on-target reads 90 read uniformity 90 median molecular coverage 500 median read coverage 15000 Tissue NGS libraries are then sequenced according to the manufacturers instructions The sequencing data of the QC passing samples are then uploaded in BAM format to the Ion Reporter Analysis Server for variant calling and annotation
Data Analysis For plasma samples variant calling is performed on Ion Reporter IR Analysis Software v510 using the Oncomine TagSeq Pan-Cancer Liquid Biopsy w20 workflows The analysis pipeline also includ signal processing base calling quality score assignment adapter trimming PCR duplicate removal and control of mapping quality Coverage metrics for each amplicon is obtained by running the Coverage Analysis Plugin software v56 Thermo Fisher Scientific Identified variants are only considered if the variant had a molecular coverage of at least three indicating that the variant is detected in three independent template molecules Finally all candidate mutations are manually reviewed using the Integrative Genomics Viewer Further annotation will be performed by Qiagen QCI platform and in-house oLIMS system
There have been small studies that examined mechanisms of resistance to anti EGFR monoclonal antibodies in mCRC using liquid biopsy A study of 37 mCRC patients who were treated with cetuximab found that 40 of them developed RAS mutations at progression 10 Another study with limited number of participants examined patients with mCRC treated with panitumumab and found that 9 out of 24 patients 38 developed KRAS mutations on treatment as a mechanism of acquired resistance to anti EGFR therapy Furthermore fewer studies with limited number of patients used liquid biopsy as a biomarker when re-challenging mCRC patients with EGFR monoclonal antibodies The majority of these studies were retrospective However one was the first prospective trial and had a similar protocol to our study It included 28 patients and reported that 52 of these patients were RAS wildtype at re-challenge with cetuximab - when these patients were exposed to and progressed on cetuximab in the first line setting This study showed that re-challenge with cetuximab significantly improved progression free survival when RAS was found to be wild type on circulating tumor DNA12 One of the limitations of this study was that a single liquid biopsy sample was done prior to re-challenge with cetuximab and hence does not display the predicted switch of the RAS target which the investigators plan to study in our trial Furthermore a more recent study protocol has been published at BMC Cancer where the investigators plan to study 120 patients and perform liquid biopsy analysis every 3 months while patients are on first line cetuximab This is to study the evolution of the RAS target and to correlate this with disease response as well as help guide therapy with EGFR inhibitors in mCRC patients However based on limited data current guidelines have not yet adopted testing using liquid biopsy and using this strategy to decide on re challenge of anti EGFR therapy in 3rd line setting or not which is the question investigators would like to answer in this study
Circulating cell free DNA cfDNA consists of small nucleic acid fragments liberated from cells by rupture necrosis or apoptosis originating from normal and deceased cells and is now increasingly being used to detect RAS and other mutations in patients with advanced colorectal cancers There is new evidence that G12C RAS mutation can be targeted with a novel anti cancer agent
the investigators aim to perform cfDNA testing on patients with advanced colorectal cancers who have no RAS mutations ie wild type and hence start on EGFR inhibitors - which is standard of care treatment pre third line therapy This will help the treating physician decide whether to give these patients with RAS wt status an anti-EGFR monoclonal antibody or standard third line therapy Regorafenib or TAS-102 These patients will be collected over an 18 month period The cfDNA test at second progression ie prior to third line systemic therapy will determine whether the subset of patients who may have developed RAS mutations after progression to first line therapy or other mutations as a mechanism of resistance with anti - EGFR monoclonal antibodies have switched their RAS status and became wild type This will support the re-challenge of EGFR inhibitors in the third line setting and has the potential of changing the colorectal cancer treatment guidelines Upon this the principle investigator will decide whether to re-challenge with anti EGFR inhibitor The investigators aim to study 60 patients in total and have 30 patients at least in the rechallenge with anti EGFR mAb group
Materials and Methods
Patients will have their standard of care SOC biopsy of tumor metastatic site to confirm diagnosis and determine RAS status Once RAS wild type and primary disease is left sided these patients will receive standard chemotherapy choices of FOLFOX FLOFIRI CapeOX XELIRI with an anti EGFR mAb cetuximab or panitumumab Upon progression of disease second line systemic chemotherapy - anti VEGF antibody will be given as per SOC Upon second progression patients will be enrolled into the study as per inclusion criteria and consent and a cfDNA blood test will be drawn and RAS status will be examined If RAS is wildtype then the investigator will decide whether to re-challenge with an anti EGFR antibody see study schema - figure 1 or give SOC third line chemotherapy Regorafenib or TAS-102
Disease assessments will be done every 8 - 12 weeks as per SOC using CT scans andor MRI and will be reported as per RECIST criteria v11
Methods for cfDNA Next Generation Sequencing NGS from CRC patients cfDNA extraction Blood samples will be collected in K2EDTA tubes BD Vacutainer Blood Collection Tubes Becton Dickinson Franklin Lakes USA and sent to the Translational Pathology Laboratory The plasma fraction will be separated from the blood cells by two consecutive rounds of centrifugation for 30 min at room temperature at 1600 g The collected plasma was aliquoted and stored at -80 C until use cfDNA is extracted from plasma volumes ranging from 04 to 55 ml using the MagMax Cell-Free Total Nucleic Acid Isolation Kit Thermo Fisher Scientific Waltham USA according to the manufacturers instructions The cfDNA quantity was assessed with the dsDNA HS assay kit by the Qubit 20 Fluorometer Thermo Fisher Scientific cfDNA quality was assessed with the Agilent Tap Station System Agilent Technologies Santa Clara USA Only cfDNA samples with a clear fragment size peak between 140-200 bp will be considered for analysis
NGS library preparation NGS libraries will prepared from 10 ng of cfDNA following the Oncomine Pan-Cancer Cell-Free Assay Thermo Fisher Scientific Our general library preparation protocol is based on a two-cycle multiplex touch-down PCR reaction with a temperature range from 64 C to 58 C which allowed to amplify target regions and introduce unique molecular identifiers The resulting tagged amplicons of around 100-140 bp length are then cleaned up using Agencourt AMPure XP Beckman Coulter Brea USA at a bead to sample ratio of 15 and purified products are eluted in 24 μl low TE buffer A second round of PCR 18 cycles will be performed in a total volume of 50 μl to amplify the purified amplicons and introduce Ion Torrent Tag-Sequencing adapters containing sample-specific barcodes The resulting library of target DNA fragments will be purified by performing a two-step cleanup using Agencourt AMPure XP Beckman Coulter at a bead to sample ratio of 115 and 10 respectively The purified libraries re then diluted 11000 and quantified by qPCR using the Ion Universal Quantitation Kit Thermo Fisher Scientific The quantified stock libraries are then diluted to 100 pM for downstream template preparation
Sequencing NGS libraries will be sequenced on an Ion S5 instrument Thermo Fisher Scientific using semiconductor sequencing technology Briefly sequencing runs are planned on the Torrent Suite Software v510 libraries are pooled and loaded on an Ion 540 chip using the Ion Chef instrument Thermo Fisher Scientific The loaded chip is then sequenced using 500 flows Raw data are processed automatically on the Torrent Server and aligned to the reference hg19 genome QC will be performed manually for each sample based on the following metrics number of reads per sample15000000 for Oncomine Pan-Cancer Cell-Free Assay libraries aries on-target reads 90 read uniformity 90 median molecular coverage 500 median read coverage 15000 Tissue NGS libraries are then sequenced according to the manufacturers instructions The sequencing data of the QC passing samples are then uploaded in BAM format to the Ion Reporter Analysis Server for variant calling and annotation
Data Analysis For plasma samples variant calling is performed on Ion Reporter IR Analysis Software v510 using the Oncomine TagSeq Pan-Cancer Liquid Biopsy w20 workflows The analysis pipeline also includ signal processing base calling quality score assignment adapter trimming PCR duplicate removal and control of mapping quality Coverage metrics for each amplicon is obtained by running the Coverage Analysis Plugin software v56 Thermo Fisher Scientific Identified variants are only considered if the variant had a molecular coverage of at least three indicating that the variant is detected in three independent template molecules Finally all candidate mutations are manually reviewed using the Integrative Genomics Viewer Further annotation will be performed by Qiagen QCI platform and in-house oLIMS system
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