Chapter III. The Study of TME in Metastatic BRAF V600E mutant CRC
Patients who met the following inclusion criteria were eligible for this study: (1)
they had pathologically confirmed de novo metastatic BRAF V600E mutated CRC,
which referred to distant metastases at diagnosis; (2) they had colectomy for CRC; and
(3) they had MSS CRC. Patients who received systemic chemotherapy or targeted
therapy before colectomy were ineligible for this study. We enrolled patients from two
medical centers: NTUH and Kaohsiung Chang Gung Memorial Hospital (KCGMH,
Kaohsiung, Taiwan). At NTUH, we searched the medical records on the BRAF mutation
examination from 2010 to 2017. The codes for BRAF mutation analyses included
000W0406 for expanded RAS+BRAF mutation, 000W0389 for KRAS and BRAF
mutations, and 000W0404 for BRAF mutation alone. Then we could identify patients
with de novo metastatic BRAF V600E mutated CRC. In addition, we included the
patients who met the inclusion/exclusion criteria from our previous cohort (2005-2013).
Finally, another cohort from KCGMH was also enrolled. We collected patients’
clinicopathological characteristics, in terms of age, sex, histology, tumor grade, disease
stage at diagnosis, primary tumor location, systemic chemotherapy, targeted therapy,
and survival time from electronic medical records. Patients were recognized to have
right-sided tumors if the primary tumors were located from the cecum to the splenic
flexure of the transverse colon. Patients had left-sided tumors if the primary tumors
were located from the descending colon to the rectum. The definition of first-line triplet
chemotherapy was the anti-cancer therapy consisting of oxaliplatin, irinotecan, and one
of the fluoropyrimidines. The first-line doublet referred to the anti-cancer therapy
consisted of one of the fluoropyrimidines and either oxaliplatin or irinotecan. This study
was approved by the Institute Review Board of NTUH and KCGMH.
BRAF V600E mutational analysis
The methods for detecting BRAF V600E mutation at NTUH include PCR and
direct sequencing (ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA).
The primer pairs (5’-TCA TAATGCTTGCTCTGATAGGA-3’ and 5’ - GGCCA
AAAATTTAATCAGTGGA-3’) covering BRAF exon 15 were used. On the other hand,
BRAF V600E mutation was identified by Roche BRAF/NRAS Mutation Test (Roche
Diagnostics, IN, USA) at KCGMH following the manufacturer’s instructions. We
performed BRAF mutation subtype classification (BM1 and BM2) by linear regression
classifier as described in a previous study79.
The method for MSI testing and the definitions of MSI-high and MSS have been
described in chapter 2.
We extracted total RNA from the FFPE samples of primary colon tumors derived
from colectomy using Qiagen’s miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA).
Macrodissection of tumor slides was performed before RNA extraction. Then we used
NanoDrop 2000 at 220–320 nm (Thermo Scientific) to measure 260/280 ratios to
determine RNA quality and quantity. Bioanalyzer (model 2100, Agilent Technologies,
Santa Clara, CA, USA) was used to measure nucleic acid fragmentation. Finally, the
gene expression was evaluated by nCounter PanCancer Immune Profiling Panel
(NanoString, Seattle, WA, USA). This panel consists of 770 genes and is widely used to
investigate TME. We processed raw data as previously described90. Gene counts for
each sample were normalized first by trimmed mean of M-values method91 and then
converted to log2-transformed counts per million later for clinical analysis.
Development of complement scores
We calculated pairwise expression correlation in complement genes according to
the similarity metric of Pearson’s correlation coefficient92. The results were regarded as
an input for hierarchical clustering. Then the complement genes were classified into
three co-expression modules, and the clustering results were shown by a heatmap
symmetrical along the diagonal line. The complement score of each sample was
determined as the average gene expression in module 2. A high complement score was
defined when the complement score was higher than the median score; in contrast, a
low complement score was defined when the complement score was lower than the
Evaluation of immune cell abundance
The immune cell abundance in the TME of each tumor was measured according to
its expression profiles. The simple average of marker genes’ expression values of each
immune cell was calculated as cell-type scores. The reference marker genes were
determined by Danaher et al.93. We also used the CIBERSORT algorithm with the
LM22 signature matrix to measure cell abundance94. The phenotypes of macrophages
(M1 and M2) were defined based on the mean expression values of gene signatures95.
The genes correlated to M1 macrophage consist of CD40, CD86, CCL5, CCR7, CXCL9,
CXCL10, CXCL11, IL1A, IL1B, IL6, IRF1, IRF5, IDO1, and KYNU, and the M2
macrophage-associated genes are CD276, CCL4, CCL13, CCL18, CCL20, CCL22,
CTSA, CTSB, CTSC, CTSD, CLEC7A, FN1, IL4R, IRF4, LYVE1, TGFB1, TGFB2,
TGFB3, TNFSF8, TNFSF12, MMP9, MMP14, MMP19, MSR1, VEGFA, VEGFB, and
The first external cohort was colon and rectal adenocarcinomas in the TCGA
dataset96. Genomic and transcriptomic data of these tumors were obtained from
Genomic Data Commons through the R package of TCGA bio-links, and these tumors’
microsatellite status was obtained according to the method described preciously97. The
second external cohort was GSE39582 dataset98. Complement scores of all these CRC
tumors from two external cohorts were calculated, and tumors were determined to have
high or low complement scores based on the abovementioned definitions.
The collected FFPE tumor tissues were cut into 5-µm sections, and then these
sections were deparaffinized by EZ prep (Ventana Medical Systems, Inc., Tucson, AZ,
USA). These sections were also pretreated with Cell Condition 1 solution for 32
minutes (Ventana Medical Systems, Inc.). We incubated the slides with anti-human C4d
(clone A24-T, DB Biotech) and used the automated Ventana Benchmark XT for 1:200
titration (Ventana Medical Systems, Inc.). Labeling was measured using the Optiview
DAB Detection Kit (Ventana Medical Systems, Inc.). All sections were counterstained
with hematoxylin in Ventana reagent, and the positive controls were the human FFPE
C4d is generated after C4 splitting during the activation of the complement
pathway. C4d is a surface-bound protein that binds to tissue near the activation site
because its covalent bond does not break spontaneously99. Thus, C4d is recognized as a
marker of complement activation. We categorized CRC into three groups based on the
percentages of C4d deposits on the tumor cell membrane. C4d-IHC 0 (weak) referred to
<1% of neoplastic cells, C4d-IHC 1 (intermediate) was defined as 1%–30% of
non-neoplastic cells, and C4d-IHC 2 (high) was defined as >30% of non-non-neoplastic cells.
Semi-quantification was evaluated by two independent pathologists.
Patients’ clinical and pathological data were summarized as percentages. Survival
analyses among groups, including PFS, OS, and progression-free interval (PFI), were
evaluated using the Kaplan-Meier method. A p-value of less than 0.05 in the log-rank
test was considered significant. We developed a Cox proportional hazards regression
model for survival analysis with the coxph function implemented in the R package. PFS
in this study was defined as the period from the date of diagnosis to the date of
radiographically confirmed progression of disease or death during first-line therapy. OS
in this study was defined as the time from diagnosis to death. PFI was specifically used
in TCGA and GSE39582 datasets to evaluate clinical outcomes96,98. PFI was defined as
the time from the date of diagnosis to the date of the first evidence of disease
progression, locoregional recurrence, new primary tumor, distant metastasis, or death.
Only variables with p < 0.1 were included in the multivariate analysis. We used
Benjamini and Hochberg multiple-testing correction method to adjust multiple tests.
The statistical analyses and visualization in this study were performed in an R
environment (version 3.6.1).