Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT04775862 |
| Other study ID # |
RC20/170/R |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
Phase 2
|
| First received |
|
| Last updated |
|
| Start date |
February 21, 2021 |
| Est. completion date |
February 2024 |
Study information
| Verified date |
February 2021 |
| Source |
National Guard Health Affairs |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial 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.
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 DNA(12). 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 i.e 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 (i.e prior to third line systemic
therapy) will determine whether the subset of patients who may have developed RAS mutation(s)
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, and/or MRI,
and will be reported as per RECIST criteria v1.1.
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 0.4 to
5.5 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 2.0 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 1.5× 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 1.15× and 1.0×,
respectively. The purified libraries re then diluted 1:1000 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™ v5.10, 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 sample>15,000,000 (for Oncomine™ Pan-Cancer Cell-Free
Assay libraries aries), on-target reads >90%, read uniformity >90%, median molecular coverage
>500×, median read coverage >15,000. Tissue NGS libraries are then sequenced according to the
manufacturer's 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 v5.10 using the Oncomine™ TagSeq Pan-Cancer Liquid Biopsy w2.0 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 v5.6 (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.
