This study is recruiting female and male patients, aged 18 and older:
Who are diagnosed with stage IV cancer (lung, colorectal, breast, liver, gastric…)
and indicated for ICI (first or second line). Concurrent chemotherapy with ICI is
allowed.
Who have not started ICI before enrollment,
Compliant with treatment protocol,
Who have no medical or psychiatric conditions or occupational, responsibilities that
may preclude compliance with the protocol,
Who consented to participate in the study.
Sample collection.
10 mL of peripheral blood (in Streck tubes) is collected for ctDNA analysis at 5
time points: pre-treatment (<10 days before ICI), during ICI every 3 months until 12
months.
6-8 sections of formalin-fixed paraffin-embedded (FFPE) tumor samples.
As part of the protocol, demographic data, height, weight, medical and family history,
and any relevant prior concomitant medication data will be recorded during follow-up
visits. All patients are to be followed for 2 years from enrollment, with CT scan/ MRI/
PET-CT imaging measured every clinical visit for 24 months.
Tumor sample processing,
Genomic DNA was isolated from FFPE and matching white blood cells (WBC) samples by the
QIAamp DNA FFPE Tissue Kit (Qiagen, USA) and the MagMAX™ DNA Multi-Sample Ultra 2.0 kit
(ThermoFisher, USA) respectively according to manufacturers' instructions. DNA
fragmentation and library preparation for both FFPE and WBC samples were performed using
the NEBNext Ultra II FS DNA library prep kit (New England Biolabs, USA). Libraries were
hybridized with predesigned probes for a gene panel of 473 targeted genes (Integrated DNA
Technologies, USA). This gene panel was curated from large public cancer databases,
high-impact cancer genomic studies, and particularly in-house database of prevalent
mutations from >10,000 cancer patients. The panel included entire exons of 473 genes,
promoter region of TERT, and selected introns of 6 common fusion genes. Massive parallel
sequencing of DNA libraries was performed on the DNBSEQ-G400 sequencer (MGI, China) with
an average depth of 500X for FFPE and 500X for WBC samples.
TMB and MSI analysis,
TMB calculation was performed using our in-house developed script to divide the total
number of eligible somatic variants by the size of the interrogated panel. An eligible
somatic variant must meet all of the following criteria: (1) pass filtering parameters of
the variant calling pipeline, (2) not likely a germline variant as filtered by the dbSNP
database, (3) locate within the coding region, (4) not a synonymous mutation, (5) have
VAF ≥ 5%, allele depth ≥ 5X, total depth ≥ 15X. The panel size was counted for the bases
within coding regions with the minimal total depth ≥ 15X. The threshold for TMB-High
(TMB-H) was determined as 10 mutations/Mb.
For MSI status, unstable microsatellite loci were detected by MSIsensor-pro (v1.2.0) in
matched tumor-normal mode. If the proportion of unstable loci among all detected
microsatellite loci was at least 20%, the sample was determined as MSI-High (MSI-H).
Tumor variant calling and ranking,
Sequencing data were processed based on best practices workflows from Genome Analysis
Tool Kit (GATK) for somatic variant calling. Specifically, reads were aligned to the
human reference genome (GRCh38) by BWA-MEM (v0.7.15). Post-alignment procedures including
sorting, marking duplicated reads, and accessing alignment quality were done by Picard
(v2.25.6). Somatic variants were called by GATK MuTect2 (v4.0.12.0) in the tumor-normal
mode for paired FFPE and WBC samples with the use of a panel of normals and the
population allele frequency from The Genome Aggregation Database (gnomAD). All filtered
variants were further assessed for their functional impact using Variant Effect Predictor
with the data from COSMIC and Clinvar databases. For mutational spectrum analysis, a
minimum Variant allele frequency (VAF) of 5% in FFPE was applied for additional
filtering. The annotated Variant Call Format (VCF) was then converted to the Mutation
Annotation File (MAF) format using vcf2maf (doi:10.5281/zenodo.593251). The MAF data were
analyzed and visualized by the 'maftools' in R package v3.4.2. All non-synonymous
alterations were ranked by our K-TrackTM scoring algorithm to identify the most potential
tumor-derived mutations to track. The top mutations unique to each patient were selected
to design bespoke multiplex PCR assays in plasma.
Plasma sample processing and multiplex PCR,
cfDNA was extracted from plasma samples using the MagMAX™ Cell-Free DNA Isolation Kit
(ThermoFisher, USA). Compatible primers were designed by Primer-BLAST software and
synthesized by PhuSa Biochem, Vietnam. cfDNA fragments carrying the selected mutation
sites were amplified in a bespoke multiplex PCR reaction containing designed primer pairs
and enzyme KAPA HiFi DNA Polymerase (Roche, USA). Additionally, cfDNA fragments were also
amplified in another multiplex PCR reaction, including primer pairs that amplify 50
target gene regions containing mutations for targeted therapy, drug resistance, and
frequent mutations in metastatic lung cancer. Amplified cfDNA fragments were indexed and
sequenced on the NextSeq 2000 system (Illumina, USA) with an average depth of 100,000X
per amplicon. Amplicons with less than 10,000X coverage were considered unsuccessful.
Plasma variant calling and ctDNA analysis,
The raw fastq data of amplicons were removed adapters with Trimmomatic (v0.39), mapped to
the human reference genome (GRCh38) using BWA-MEM (v0.7.15), sorted and marked duplicates
using Picard (v2.25.6). Variant calling was performed using mpileup from Samtools
(v1.11). A plasma sample that had at least one mutation with VAF above 0.05% was defined
as ctDNA positive.
