ORIGINAL ARTICLE

Genetic Intratumor Heterogeneity Remodels the

Immune Microenvironment and Induces Immune
Evasion in Brain Metastasis of Lung Cancer
Xin Wang, MD,a Hua Bai, MD,b Jiyang Zhang, PhD,i Zhijie Wang, MD,b
Jianchun Duan, MD,b Hongqing Cai, MD,f Zheng Cao, MD,c Qingtang Lin, MD, PhD,g
Xiaosheng Ding, MD,h Yiting Sun, MD,b Wei Zhang, PhD,i Xiaoya Xu, MS,i
Hao Chen, PhD,i Dadong Zhang, PhD,i Xiaoli Feng, MD,c Jinghai Wan, MD,f
Jianjun Zhang, MD, PhD,d,e Jie He, MD, PhD,a Jie Wang, MD, PhDb,*
a
Department of Thoracic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
b
State Key Laboratory of Molecular Oncology, Department of Medical Oncology, National Cancer Center/National Clinical
Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing, China
c
Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing, China
d
Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA
e
Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston,
TX, USA
f
Department of Neurosurgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
g
Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China
h
Department of Oncology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China
i
Department of Translational Medicine, 3D Medicines Inc., Shanghai, China

Received 6 June 2023; revised 18 August 2023; accepted 7 September 2023

Available online - XXX

ABSTRACT Conclusions: Our findings provide novel insights into the

metastatic process and immune escape mechanisms of brain
Introduction: Brain metastasis, with the highest incidence in
metastasis and pave the way for precise immunotherapeutic
patients with lung cancer, significantly worsens prognosis and
strategies for patients with lung cancer with brain metastasis.
poses challenges to clinical management. To date, how brain
metastasis evades immune elimination remains unknown.
Methods: Whole-exome sequencing and RNA sequencing
were performed on 30 matched brain metastasis, pri-
mary lung adenocarcinoma, and normal tissues. Data
from The Cancer Genome Atlas primary lung adeno-
carcinoma cohort, including multiplex immunofluores-
cence, were used to support the findings of
bioinformatics analysis.
Results: Our study highlights the key role of intratumor
heterogeneity of genomic alterations in the metastasis
process, mainly caused by homologous recombination
deficiency or other somatic copy number alteration–
associated mutation mechanisms, leading to increased
genomic instability and genomic complexity. We further
proposed a selection model of brain metastatic evolution in
which intratumor heterogeneity drives immune remodeling,
leading to immune escape through different mechanisms
under local immune pressure.
*Corresponding author.
Drs. X. Wang, H. Bai, and J.Y. Zhang contributed equally to this paper.
Drs. J. He and J. Wang contributed equally as co-senior authors.
Disclosure: Dr. H. Chen is a former employee of 3D Medicines Inc. Drs.
J.Y. Zhang, W. Zhang, X.Y. Xu, and D.D. Zhang are current employees
of 3D Medicines Inc. The remaining authors declare no conflict of
interest.
Address for correspondence: Jie Wang, MD, PhD, State Key Laboratory
of Molecular Oncology, Department of Medical Oncology, National
Cancer Center/National Clinical Research Center for Cancer/Cancer
Hospital, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China, 17 Pan-jia-yuan South Lane, Chaoyang
District, Beijing 100021, People’s Republic of China. E-mail: zlhuxi@
163.com
ª 2023 International Association for the Study of Lung Cancer.
Published by Elsevier Inc. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
ISSN: 1556-0864
https://doi.org/10.1016/j.jtho.2023.09.276

Journal of Thoracic Oncology Vol. - No. -: -–-

2 Wang et al
 2023 International Association for the Study of Lung
Cancer. Published by Elsevier Inc. This is an open access
article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Brain metastasis; Patient-paired samples; Intra-
tumor heterogeneity; Immune editing; Precise
immunotherapy
Introduction
The high morbidity and mortality of brain metastasis
from lung cancer presents an enormous obstacle for
clinical management and treatment.1–3 Accumulating ev-
idence has revealed that brain metastasis is an extremely
complex process that not only involves genetic alterations
but also contributes to the tumor immune microenvi-
ronment (TIME).4,5 The evolution direction of tumor
cloning is determined by the internal characteristics of
tumor cells and the Darwinian selection rule mediated by
external immune stress.6–8 Nevertheless, the interplay
between the TIME and genomic alterations during brain
metastasis is not well understood and requires more
extensive mechanistic analyses.
Paired analysis of primary metastatic samples has the
potential to distinguish events that occur before and
after dissemination and infer whether any events are
recurrently selected in metastatic clones.9–12 The
exploration of driver heterogeneity between primary
tumors and their matched metastases has revealed that
most driver alterations tend to be clonal, occurring on
the trunk of a tumor’s phylogenetic tree and thus
necessarily in every metastatic cancer cell.13–17 Theo-
retically, metastasis is an extremely complex process in
which tumor cells leave their primary site, disseminate
into a secondary site, adapt to the new environment, and
proliferate by evading immune surveillance.18–20
Therefore, metastasis is a process that has the poten-
tial to introduce considerable heterogeneity in metasta-
tic tumors.12,21,22
Although a few recent studies have addressed the
role of the immune system in brain metastatic hetero-
geneity,23–26 brain metastatic seeding and evolutionary
clonal dynamics under immune pressure remain elusive
in the absence of a multiomics cohort study. Other
studies, however, have revealed the effects of the TIME
on tumor clonal heterogeneity in mouse models27 and its
interaction with genetic alterations which has an impact
on patient survival.28 Consequently, an improved inves-
tigation of the processes leading to brain metastatic in-
vasion and development is needed to devise novel
treatment paradigms for brain metastatic tumors.
We hypothesized that brain metastatic development
and the clonal evolution of tumor cells underlie
Journal of Thoracic Oncology Vol. - No. -
Darwinian selection, mediated by both tumor-intrinsic
characteristics and extrinsic host factors, particularly
immune pressure. To explore how tumor microenvi-
ronment selection pressures affect brain metastatic
evolution, we generated genomic and transcriptional data
on paired primary lung tumors and brain metastases from
22 patients with lung adenocarcinoma (LUAD) (Fig. 1A
and B). We characterized brain metastatic evolution as
intratumor heterogeneity (ITH)–based clone selection
under the immune microenvironment and elucidated the
relationships between immune editing and ITH and their
impact on clinical prognosis. We also validated these
findings in an external cohort of eight pairs of primary
LUAD (LP) and brain metastasis (BM) samples using
whole-exome sequencing (WES) and RNA sequencing
(RNA-seq), including data from The Cancer Genome Atlas
primary LUAD cohort. Intriguingly, we developed a par-
allel model of brain metastatic evolution under local im-
mune pressure that led to immune escape through
different mechanisms (Fig. 1C).
Materials and Methods
Patients and Specimens
This study protocol was reviewed and approved by
the institutional ethnic committee in the Cancer Hospital,
Chinese Academy of Medical Sciences (NCC2016G-074).
All patients were consented about human specimen
collection. We reviewed patients with lung cancer with
brain metastasis, who had surgically resected primary
lung tumors and matched brain metastases (without
steroid hormone treatment before surgery) between
2010 and 2019. After extensive quality control, 30 pa-
tients (22 patients in NCC [National Cancer Center/
Cancer Hospital Chinese Academy of Medical Sciences]
cohort and 8 patients in external validation cohort) with
complete medical records and sufficient specimen were
included for subsequent sequencing study. Formalin-
fixed, paraffin-embedded (FFPE) specimens of NSCLC
tumor, matched normal tissue, and brain metastasis
were collected. Hematoxylin and eosin–stained sections
from all tumor samples were subjected for confirmation
independently by two certified pathologists (X.L. Feng
and Z. Cao) that the tumor specimen was histologically
consistent with metastatic NSCLC and that the matched
normal tissue specimen contained no tumor cells. Tu-
mors were assessed to have greater than or equal to
60% tumor cell purity and less than 20% necrosis.
Data Collection and Follow-Up
Electronic medical records were reviewed to collect
information on clinicopathologic characteristics, which
included patient sex, age, smoking history, pathologic
subtype, pathologic stage, size of primary lung lesion,
--- 2023 Intratumor Heterogeneity of Brain Metastasis 3

Figure 1. Overview of the study. (A) Demographic and clinical characteristics of the NCC cohort (n ¼ 22) and external cohort
(n ¼ 8) in the study, including diagnosis, tumor stage, and smoking status. (B) Workflow of brain metastasis, primary tumor,
and normal tissue collection, processing, and sequencing. (C) Modeling the brain metastatic process in lung cancer: brain
metastatic development and clonal evolution of tumor cells underlie Darwinian selection mediated both by tumor-intrinsic
characteristics and extrinsic immune pressure. In this context, subclones lead to ITH from SCNA or HRD, which give a se-
lective advantage and begin a selective sweep of the tumor. Subclones containing neoantigens that stimulate the anticancer
immune response are eliminated by active T cells. Meanwhile, an exclusively genomic approach would fail to identify the
functional genomic alterations shaping tumor evolution under local immune pressure leading to immune escape through
different mechanisms. HRD, homologous recombination deficiency; ITH, intratumor heterogeneity; NCC, National Cancer
Center/Cancer Hospital Chinese Academy of Medical Sciences; SCNA, somatic copy number alteration.
4 Wang et al
date of initial diagnosis, date of lobectomy, date of
complete brain metastasis resection, treatment after lo-
bectomy, treatment after BM, and date of death or last
date of follow-up. The clinical information of all 22 pa-
tients is listed in Supplementary Table 1.
DNA and RNA Extraction and Quality Control
We extracted DNA and RNA from FFPE specimens
using the DNA FFPE Tissue Kit and RNeasy FFPE Kit
(Qiagen) according to the protocol’s instructions. The
amount of extracted DNA was determined by Qubit
(Thermo Fisher), and the integrity of DNA was deter-
mined by agarose electrophoresis. The quality of RNA
was determined by a 2100 Bioanalyzer (Agilent) with
DV200 (percentage of RNA fragments >200 nt fragment
distribution value) and quantified using a NanoDrop
system (Thermo Scientific).
Whole-Exome Sequencing
For WES, 500 ng of genomic DNA was used for li-
brary preparation. A sequencing library was constructed
by the SureSelect Human All Exon Kit (V5, Agilent)
following the manufacturer’s instructions. After quality
control, libraries were sequenced on an Illumina Nova-
Seq platform. Validated sequencing data were mapped to
the human reference genome hg19 by the Burrows-
Wheeler Aligner (0.7.12-r1044).
RNA Sequencing
RNA purification, reverse transcription, library con-
struction, and sequencing were performed according to the
manufacturer’s instructions (Illumina). Captured coding re-
gions of the transcriptome from total RNA were prepared
using the TruSeq RNA Exome Library Preparation Kit. RNA
input for library construction was determined by the quality
of RNA. Generally, 20 ng of RNA was recommended for
high-quality RNA, 20 to 40 ng of RNA for medium-quality
RNA, and 40 to 100 ng of total RNA for low-quality RNA.
Then, complementary DNA was generated from the input
RNA fragments using random priming during first- and
second-strand synthesis, and sequencing adapters were
ligated to the resulting double-stranded complementary
DNA fragments. Coding regions of the transcriptome were
then captured from this library using sequence-specific
probes to create the final library. After library construc-
tion, a Qubit 2.0 fluorometer double-stranded DNA HS assay
(Thermo Fisher Scientific) was used to quantify the con-
centration of the resulting sequencing libraries, whereas the
size distribution was analyzed using an Agilent BioAnalyzer
2100 (Agilent). Sequencing was performed using an Illu-
mina system following the manufacturer’s instructions for
2  150 paired-end sequencing.
Journal of Thoracic Oncology Vol. - No. -
See Supplementary Files for more information of the
methods.
Results
Description of the Sequencing Cohort
To comprehensively explore the interplay between
intratumor genetic heterogeneity and selection pres-
sures in immune infiltration, we recruited 22 patients
with LUAD with brain metastasis between January 2010
and May 2019 as NCC cohort who underwent surgery. In
addition, eight patients (four patients with LUAD and
four patients with lung squamous carcinoma) were
included for external validation (Supplementary Fig. 1A).
Paired normal lung tissues, LP tissues, and BM tissues
were collected from each patient (Fig. 1A), including five
patients with synchronous brain metastases (diagnosed
at the same time as the primary lung tumor) and 25
patients with metachronous brain metastases (diag-
nosed during the disease course). Hematoxylin and
eosin–stained images of paired primary LUAD and brain
metastasis tissues of the 14 patients are presented in
Supplementary Fig. 2. The median follow-up time for this
cohort was 40 months (5.4–132.5 mo), and 40% of the
patients died during the follow-up. The clinical data are
summarized in Supplementary Table 1.
For each sample, we performed multiple genomic and
infiltrating immune cell assays, including WES and
transcriptome profiling (RNA-seq), totaling 130
sequencing profiles in the NCC cohort (Fig. 1B). Tumor
and normal tissue samples were sequenced at a median
of 195-fold and 134-fold coverage and across exome
capture loci. All samples were processed within a uni-
form bioinformatics pipeline to identify somatic single-
nucleotide variants (sSNVs), insertions/deletions, mi-
crosatellite instability (MSI), mutation signatures, ho-
mologous recombination deficiency (HRD), somatic copy
number (CN) alterations (SCNAs), gene expression
levels, TIME, and neoantigen burden (Supplementary
Fig. 1B).
Genomic Landscapes of Paired Primary Tumors
and Brain Metastases
We first looked for evidence of enrichment of func-
tional driver mutations in brain metastasis tumors. To
systematically evaluate the mutational spectrum, we
defined functional (“driverness”) mutations with onco-
genic, therapeutic, or prognostic implications
(Supplementary Table 2) in the TARGET13,29 database.
The top two drivers were EGFR (mutation frequency LPs
versus BMs, 68% versus 73%, respectively) and TP53
(mutation frequency LPs versus BMs, 64% versus 73%,
respectively). In total, 120 functional driver sSNVs or
insertions/deletions were detected across paired
--- 2023 Intratumor Heterogeneity of Brain Metastasis 5

A B

F G

Figure 2. Genomic landscapes in paired primary tumors and brain metastases. (A) Landscape of somatic mutations of driver
genes in the 22 paired LP and BM samples in this study. Top: The number of somatic mutations in each sample. Middle: The
matrix of mutations in a selection of frequently mutated genes. Only genes that were present in more than two cases (in
either primary lung tumors or brain metastases) are found. The mutation frequencies of these genes in our cohort are found
on the right. Bottom: Mutation types are represented by the colors indicated. (B) Total TMB count (defined as the number of
6 Wang et al Journal of Thoracic Oncology Vol. - No. -

primary tumors and brain metastases (Fig. 2A and
Supplementary Table 2). Nevertheless, we failed to find
that either drivers or nondrivers were significantly
enriched in BMs (Supplementary Table 3). These results
are consistent with those of previous studies9,26,30 that
revealed high driver gene concordance between primary
tumors and metastases with few metastasis-private
driver mutations identified, suggesting that the
genomic drivers required for brain metastasis often
occur early in the primary lung tumor, which supports
the late dissemination model.9
Next, we computed tumor genetic features, such as
tumor mutation burden (TMB) and somatic MSI index,
using MSIsensor231 (Supplementary Table 4; Methods).
Both TMB (paired Wilcoxon ranked sum test p > 0.05;
Fig. 2B) and MSI status (all samples were MSI negative,
MSI score < 20%; paired Wilcoxon ranked sum test p >
0.05; Fig. 2C) were not significantly different between LP
and BM pairs.
To further determine which mutational processes
are involved in brain metastasis, we deconvoluted
mutational signatures using deconstructSigs32 on the
basis of a predefined mutational spectrum of 30
COSMIC signatures. BMs had an increase in the S3
signature (HRD) and a decrease in the S1 signature
(aging) compared with LPs (Wilcoxon ranked sum
test p < 0.01; Fig. 2D and Supplementary Tables 5
and 6). To further investigate the dynamics of BM
mutational signatures, we separated all mutations
into the following three categories: mutations found
only in BMs (BMs only), mutations shared by BMs
and LPs (shared mutations), and mutations occurring
only in LPs (LPs only) (Fig. 2D and Supplementary
Table 7; Methods). The shared mutations represent
early and ancestry mutations. On a relative scale, we
observed a shift from more indolent age-related
mutagenesis in primary lung tumors toward more
HRD-driven processes in brain metastatic tumors
(Fig. 2D).
To reaffirm the HRD status accurately, scarHRD33
was used to calculate the combined HRD score, which
was defined as the sum of the loss of heterozygosity
(LOH), number of large-scale transitions, and telomeric
allelic imbalance scores on the basis of WES data
(Methods and Supplementary Table 4). Compared with
LPs, BMs had significantly higher HRD scores (paired
Wilcoxon ranked sum test p ¼ 7.2E-03; Fig. 2E) with an
average variation (fold-change) of 1.61, matching the
observed difference in the above-mentioned mutation
signature analysis. To further confirm the findings, we
obtained in the validation cohort eight paired lung can-
cer and brain metastatic tumors. In keeping with results
from the NCC cohorts, we observed a significant increase
of HRD score in BMs (paired Wilcoxon rank sum test, p ¼
0.030; Supplementary Fig. 3A). Comparing homologous
recombination repair (HR) genes (Supplementary
Fig. 3B) CNA events between BMs and LPs
(Supplementary Fig. 3C), we found a significantly
elevated degree of SCNA events of HR gene in BMs
(Fisher’s test, p ¼ 6.13E-05; Supplementary Fig. 3C).
Furthermore, HRD score was higher in BMs with HR
genes CNA than that without HR genes CNA
(Supplementary Fig. 3D). When HRD score in BMs was
higher than in LPs (HRD ratio > 1), TMB was also higher
in BMs than in LPs (Supplementary Fig. 3E and F). It is
suggested that HRD enrichment by elevating genomic
instability was significantly associated with BM
development.

nonsynonymous mutations) in BMs and LPs. (C) MSI score in BMs and LPs. Both TMB and MSI scores were not significantly
different between BMs and LPs. (D) Mutational signatures in LPs and matched BMs. Left: Major mutational signatures in LPs
and BMs. In LPs and BMs on the basis of the 30 predefined COSMIC signatures, the top seven signatures in BMs were presented.
Middle: Aging and HRD signatures were significantly different between BMs and LPs. The aging signature was significantly
decreased in BMs compared with LPs (paired Wilcoxon ranked sum test, p ¼ 0.0022). The HRD signature was significantly
elevated in BMs compared with LPs (paired Wilcoxon ranked sum test, p ¼ 0.0061). Right: All mutations were classified into
three groups: LP only, shared, and BM only, and mutational signatures were extracted in each group. (E) HRD scores (defined
as the sum of LOH score, LST score, and TAI score) in LPs and matched BMs. BMs data are on the X-axis and LPs data are on the
Y-axis. The HRD score was significantly higher in BMs than in LPs (paired Wilcoxon ranked sum test p ¼ 7.2E-0.3). The LOH
score was significantly higher in BMs than in LPs (paired Wilcoxon ranked sum test p ¼ 5.1E-03). The LST score was signifi-
cantly higher in BMs than in LPs (paired Wilcoxon ranked sum test p ¼ 1.2E-02). The TAI score was not significantly different
between BMs and LPs (paired Wilcoxon ranked sum test p ¼ 0.12). (F) SCNA burden in LPs and BMs. The SCNA burden in BMs
was significantly higher than that in LPs (paired Wilcoxon ranked sum test p ¼ 3.8E-5). (G) KEGG pathway enrichment analysis
genes with CNA which only occurred in BMs. Left: The top 10 pathways from the KEGG enrichment analysis ranked by
p.adjust-value, found as a bar chart. Right: A bar plot revealed the numbers of genes with CNA which only occurred in BMs
which in TGF-b and HR pathway, respectively. X-axis: Number of genes related to the enriched KEGG pathway, Y-axis:
functional pathways. The color of the bar denotes -log 10 (p.adjust-value). The p values have been adjusted by Benjamini-
Hochberg method. Signaling pathways... stem cells is “Signaling pathways regulating pluripotency of stem cells.” BM, brain
metastasis; CNA, copy number alteration; HR, homologous recombination; HRD, homologous recombination deficiency; LOH,
loss of heterozygosity; LP, primary lung adenocarcinoma; LST, large-scale state transition; MSI, microsatellite instability;
SCNA, somatic copy number alteration; TAI, telomeric allelic imbalance; TGF, transforming growth factor; TMB, tumor
mutation burden.
--- 2023
SCNAs are often associated with genomic insta-
bility,34,35 are widely spread in human cancers, and drive
the evolution of BM-LUAD.36 At the whole-genome level,
the SCNA burden (defined as the total number of genes
with CN gain or loss per sample) was significantly higher
in BMs than in LPs by the established algorithm
Sequenza37 (Wilcoxon ranked sum test p ¼ 3.8E-05;
Fig. 2F and Supplementary Table 4), regardless of the
SCNA gain burden (Wilcoxon ranked sum test p ¼ 1.2E-03;
Supplementary Fig. 4A) and SCNA loss burden (Wilcoxon
ranked sum test p ¼ 8.9E-03; Supplementary Fig. 4B).
Next, we confirmed the elevation of SCNA burden in BMs
using the external validation data set (Supplementary
Fig. 4C). We also obtained the concordant results that
SCNA burden elevated in BMs at the DNA segment level by
the other algorithm TitanCNA38 (Pearson’s correlation
analysis p ¼ 6.14E-06, R ¼ 0.62; Supplementary Fig. 4D
and E) (Methods), which validated that EGFR and SMAD4
were frequently involved in genomic amplifications
and deletions, respectively, in a brain metastasis
(Supplementary Fig. 4F and G). Between paired LPs and
BMs, the fold-change of the HRD score and the fold-change
of the SCNA burden were weakly to moderately correlated
with each other (Spearman’s correlation analysis p > 0.05;
Supplementary Fig. 4H). Next, we performed KEGG
pathway enrichment analyses on the genes with CNA
which only occurred in BMs and found that those insta-
bility genes were mainly enriched in tumorigenic path-
ways, such as Rap1 signaling, Ras signaling, MAPK
signaling, transforming growth factor (TGF)-b signaling,
and HR pathways (Fig. 2G), associated with the immuno-
suppression, angiogenesis, and metastasis of tumors. On
the basis of these results, it is concluded that BMs had a
higher SCNA burden than paired LPs, which represents
increased genomic instability and complexity in BMs.
Overall, the above-mentioned evidence supports a het-
erogeneous genomic landscape in which brain metastasis
arises from the primary tumor through the continual
accumulation of genomic instability rather than the intro-
duction of novel driver SNVs.
Genetic Intratumor Heterogeneity of Paired
Primary Tumors and Brain Metastases
Genetic intratumor heterogeneity, describing the
diversity within individual tumors,39 is the cell-to-cell
variation in cancer evolution40 and arises through
genomic instability by different mechanisms, such as
subclonal driver mutations and CNAs.41 It can be
assessed by the mutant-allele tumor heterogeneity
score (which we defined as the “ITH score”), a metric
inferred from the somatic mutation variant allele fre-
quency distribution27,42–44 (Methods). We observed
that BMs had significantly higher ITH scores than
Intratumor Heterogeneity of Brain Metastasis 7
paired LPs (paired Wilcoxon ranked sum test, p ¼ 2.0E-
04; Fig. 3A), with an average variation (fold-change) of
1.44. The ratio of the BM ITH score to the paired LP ITH
score was greater than 1 in 77% (17 of 22) of the
patients, with the remaining patients (five of 22) hav-
ing values greater than 0.85 (Supplementary Table 4).
The above-mentioned ITH discrepancies were
confirmed by another established method, PyClone45
(Methods), which defines ITH as a tumor clone num-
ber: BMs had a significantly higher clone number than
paired LPs (paired Wilcoxon ranked sum test, p ¼ 8.8E-
03; Fig. 3B). The discrepancy in the ITH score in BMs
and paired LPs remained consistent in the external
validation cohort (paired Wilcoxon ranked sum test,
p ¼ 1.6E-02; Supplementary Fig. 5A and
Supplementary Table 8). The above-mentioned data
suggest that high ITH plays a distinct internal role in
brain metastasis, including changes during the evolu-
tionary process of BM.
In addition, the ITH score of BMs was not signifi-
cantly correlated with the ITH score of LPs (Pearson’s
correlation analysis p > 0.05; Supplementary Fig. 5B).
Furthermore, no correlation was observed between ITH
score and TMB (Spearman’s correlation analysis p >
0.05; Supplementary Fig. 5C). No differences were
observed in the ITH score, HRD score, or SCNA burden
between the synchronous and metachronous brain
metastasis groups (Wilcoxon ranked sum test p > 0.05;
Supplementary Fig. 6A–C). The above-mentioned results
reveal that ITH in brain metastatic lesions cannot be
inferred from the characteristics of LP, so sequencing
metastatic samples would be required to elucidate the
unique characteristics of BM, whose importance was
frequently omitted in previous studies.
To further explore the source of intratumor genetic
heterogeneity that arises through the continual accu-
mulation of DNA damage during metastasis evolution,
we compared SCNA alterations between LPs and BMs
and their correlation with ITH score. As expected,
repair system damage, including HRD, which dampens
genomic stability accompanied by more SCNAs, was
frequently observed in BMs (77.3% of BMs, 17 of 22)
(Fig. 3C). We also found that the ITH score was strongly
and positively correlated with SCNA burden (Pearson’s
correlation p ¼ 3.27E-07, R ¼ 0.72; Fig. 3D) and HRD
score (Pearson’s correlation p ¼ 2.2E-03, R ¼ 0.44;
Fig. 3E). Overall, these data suggest that the ITH vari-
ation between LP and paired BM tissues might be
attributed to SCNA accumulation, predominantly the
result of genomic instability caused by HRD and other
DNA repair pathway alteration. These SCNAs were
established late during metastatic tumor evolution and
persisted through clone selection.
8 Wang et al Journal of Thoracic Oncology Vol. - No. -

A B

D E

Figure 3. Intratumor genetic heterogeneity of paired primary lung tumors and brain metastases. (A) Comparison of ITH
scores between pairwise LPs and BMs. The score represents a measure of the level of intratumor heterogeneity and was
calculated for each tumor as described in the Methods section. The ITH score was significantly higher in BMs than in paired LPs
(paired Wilcoxon ranked sum test p ¼ 0.0002). n ¼ 22 BM/LP pairs. (B) Clone number distribution as predicted by PyClone
within each group. The number of clones represents a measure of the level of ITH and was calculated for each tumor as
--- 2023 Intratumor Heterogeneity of Brain Metastasis 9

Heterogeneity of the TIME and Interaction
Between Intratumor Genetic Heterogeneity and
Immune Infiltration
Clonal diversity facilitates tumor evolution, ultimately
shaping genomic features and temporal alterations of
compartments in the TIME as a fertile soil. Immune
infiltration, as a critical component of the TIME, plays a
key role in cancer progression. To characterize the im-
mune infiltrate configuration of paired tumors, we first
inferred the abundance of 28 subpopulations of infil-
trating immune cells from RNA-seq data (Supplementary
Table 9; Methods) using the single-sample gene set
enrichment analysis algorithm46 (Supplementary
Table 10; Methods). Unsupervised hierarchical clus-
tering revealed two distinct immune clusters for LPs and
BMs, which corresponded to high and low levels of im-
mune infiltration, respectively (Fig. 4A). To further assess
immune infiltration, we used a normalized score called
the “immune infiltration score” to assess the activation
status of the antitumor ability of infiltrating cells46
(Methods). BMs exhibited significantly lower immune
infiltration scores than LPs (paired Wilcoxon ranked sum
test p ¼ 0.0085; Fig. 4B), in line with the results calcu-
lated by two other algorithms, including “GEP”47 (a T cell-
inflamed gene expression profile) (paired Wilcoxon
ranked sum test p ¼ 1.5E-03; Supplementary Fig. 7A and
Supplementary Table 10) and “ImSig”48 (paired Wilcoxon
ranked sum test p ¼ 6.0E-03, Supplementary Fig. 7B and
Supplementary Table 10). CIBERSORT49 data suggested
that protumor M2 macrophages were significantly higher
in brain metastases (paired Wilcoxon ranked sum test p ¼
2.9E-02, Supplementary Fig. 7C and Supplementary
Table 11). We also observed that vascular epithelial
growth factor-A was highly expressed in the BM tumor
samples, which are known to contribute to neoangio-
genesis, anti-inflammatory cytokine milieu, and estab-
lishment of the immunosuppressive state50 (paired t test
p ¼ 2.6E-02; Supplementary Fig. 7D). To validate whether
the discrepancy of the immune infiltrations between LPs
and BMs was also reflected by tumor immune cell density,
we analyzed the densities of the immune cells within
paired tumor tissues using multiplex immunofluores-
cence (mIF) (Supplementary Table 12). Indeed, compared
with LPs, we observed low levels of immune infiltration,
decreased densities of CD3þ, CD4þ, and CD8þ T cells,
and no density changes of programmed death-ligand 1
(PD-L1)þ cells in BMs, consistent with RNA-seq data
(Fig. 4C–E and Supplementary Fig. 7E–H). We also found a
positive correlation between immune score calculated by
RNA-seq data and CD3þ, CD4þ, and CD8þ T cell den-
sities derived from mIF data (Pearson’s correlation p <
0.05, Supplementary Fig. 7I–K). Above all, BMs had lower
immune cell infiltration than paired LPs, suggesting an
inactive or “cold” immune microenvironment.23,26 To
determine the interaction between genomic features and
the TIME, distances on the basis of ITH and immune
differences were determined for all paired tumors from
the same patients7 (Methods). We observed a significant
positive correlation between the two pairwise distance
measures (Spearman’s correlation p ¼ 2.1E-02, R ¼ 0.52;
Fig. 4F [left]), which reflects the interaction between
genomic features and the TIME. To further explore the
relationship between ITH and immune infiltration, we
calculated ITH scores and immune infiltration scores for
all pairwise samples (Methods). Notably, a strongly
negative correlation was observed between the immune
infiltration and ITH scores (Pearson’s correlation p ¼
2.3E-06, R ¼ 0.64; Fig. 4F [right], GEP algorithm, Pear-
son’s correlation p ¼ 4.55E-05, R ¼ 0.57;
Supplementary Fig. 8A, Imsig algorithm, Pearson’s corre-
lation p ¼ 3.89E-06, R ¼ 0.63; Supplementary Fig. 8B),
which was validated in the external cohort (Pearson’s
correlation p ¼ 3.1E-02, R ¼ 0.62; Supplementary
Fig. 8C and Supplementary Table 13) and The Cancer
Genome Atlas database (Pearson’s correlation p ¼ 6.42E-
10, R ¼ 0.26, Supplementary Fig. 8D). In addition,
densities of infiltrating activated CD8þ T cells by mIF
were negatively correlated with ITH score (Fig. 4G). Our
results, to the best of our knowledge, reveal for the first
time that ITH is negatively correlated with the level
of tumor immune infiltration and that TME remodeling in
BM evolution has important implications in tumor
development.
Genetic Intratumor Heterogeneity and Immune
Editing of Brain Metastasis
The previous section actually leaves us an intriguing
question: How is the cold immune microenvironment in
BM established during immunoediting? According to the
immune editing theory, we hypothesized that in

described in the Methods section. The clone number was significantly higher in BMs than in LPs (paired Wilcoxon ranked sum
test p ¼ 0.0088). (C) Scattergram reveals changes in the SCNA burden and HRD score in each BM compared with its paired LP.
BMs of approximately 90% (20 of 22) had an increased SCNA burden or HRD score compared with paired LPs. Red points
indicate SCNA burden or HRD score increases and SCNA burden less than or equal to median value in BMs, triangles indicate
SCNA burden greater than median value in BMs, and black points indicate that neither SCNA burden and HRD score increases.
(D) Positive correlation between SCNA burden and ITH score (Pearson’s correlation analysis, p ¼ 3.3E-07). (E) Positive cor-
relation between HRD score and ITH score (Pearson’s correlation analysis, p ¼ 0.0022). BM, brain metastasis; HRD, homol-
ogous recombination deficiency; ITH, intratumor heterogeneity; LP, primary lung adenocarcinoma; SCNA, somatic copy
number alteration.
10 Wang et al Journal of Thoracic Oncology Vol. - No. -

A B

C D

F G

Figure 4. Heterogeneity of the TIME across paired primary lung tumors and brain metastases. (A) The relative fraction of
tumor-infiltrating lymphocyte cells using the ssGSEA algorithm with available RNA-sequencing data in the LP and BM groups.
The relative infiltration of each cell type is normalized into a z-score. (B) The change in immune cell infiltration score
(Methods) is found between each BM and paired LP sample. Dot plots reveal that the immune score was significantly lower
in the BM (“cold tumor”) (red) than in the LP groups (“hot tumor”) (blue) in our study (paired Wilcoxon ranked sum test
--- 2023 Intratumor Heterogeneity of Brain Metastasis 11

metastatic lesions, highly immunogenic tumor clones
would be eliminated under immune pressure and only
clones or subclones with neoantigen depletion could
survive. This Darwinian selection would eventually
lower immune infiltration and be more efficient in BM
under high ITH, which provides many subclones with
neoantigen depletion for selection, so the immune
microenvironment in BMs would be cold compared with
that in LPs.
Because neoantigen depletion can occur at the DNA
level (through events such as CN loss) or at the RNA
level (through the suppression of transcripts that
contain neoantigens),7 to prove the above-mentioned
hypothesis, both levels were explored. We discovered
neoantigen depletion at the DNA level through CN loss.
BMs (12 of 22, range: 0%–30% of neoantigens; Fig. 5A)
had more evidence (Fisher’s extract test p ¼ 2.8E-03;
Supplementary Fig. 9A) for neoantigen loss events due
to CN loss events than paired LPs (two of 22, range:
0%–0.03% of neoantigens; Fig. 5A). Alternatively, to
further investigate neoantigen depletion at the RNA
level in BMs, we first discovered that the expression
level of genes associated with neoantigen was lower in
CN loss regions (p ¼ 0.013; Fig. 5B) and next compared
the expected (ignoring the expression-based filter) and
observed (the expression-based filter or on a region of
CN loss) numbers of neoantigens present between the
two groups51 (Supplementary Table 13; Methods). We
found that the expected number of neoantigens and
observed neoantigens were highly concordant be-
tween LPs and BMs (paired Wilcoxon ranked sum test
p > 0.05; Fig. 5C). Then, we used the observed-to-
expected ratio of neoantigens to further estimate
neoantigen depletion.51 The ratio was near/close to 0,
suggesting an increase in DNA/RNA neoantigen
depletion capacity and immune editing. As expected,
BMs had a significantly lower observed-to-expected
ratio of neoantigens (an increase in immunoediting)
than LPs (paired Wilcoxon ranked sum test p ¼ 3.05E-
04; Fig. 5C). Generally, immune editing events (i.e.,
immune depletion) increased in BMs due to increasing
CN loss at the DNA level and decreasing observed
neoantigen expression at the RNA level, which actually
led to the low BM immune infiltration found in the
previous section.
Next, we investigated the interplay between immune
editing and intratumor genetic heterogeneity. In both
BMs and LPs, high-ITH tumors had significantly
increased immunoediting events (Spearman’s correla-
tion p ¼ 1.56E-04, R ¼ 0.54; Fig. 5D), which negatively
correlated with immune score derived from gene
expression profiling (Spearman’s correlation p ¼ 0.049,
R ¼ 0.3; Fig. 5E). To further validate immunoediting
status of tumors, we found a positive correlation be-
tween CD4þ and CD8þ T cell density by mIF and
observed-to-expected ratio of neoantigens (Spearman’s
correlation p ¼ 6.41E-03, R ¼ 0.63; Fig. 5F). These re-
sults support that immune editing events occurring
during the brain metastasis process are associated with
ITH and that elevated temporal ITH correlates with the
depletion of neoantigens at both the RNA and DNA
levels, proving the hypothesis proposed at the beginning
of this section. In summary, we delineated the links
among genetic ITH, immunoediting, and tumor escape
mechanisms in evolutionary processes across primary
lung tumors and brain metastases.
ITH-Dependent Immune Evasion Mechanism in
Brain Metastases
During tumor metastasis, cancer cells harness a vari-
ety of mechanisms to evade recognition and elimination

p ¼ 0.00018). (C) Clustering based on the level of estimated immune infiltrate. Each row represents immune cell populations,
as estimated by the method used by mIF, including the density of CD3þ T cells, CD4þ T cells, CD8þ effector T cells,
CD68þCD163 macrophage cells M1, CD68þCD163þ macrophage cells M2, CD20þ B cells, CD56þ NK cells, and Foxp3þ CD4þ
T regulatory cells in tumor tissues. The relative infiltration of each cell type is normalized into a z-score. (D) Comparisons of
immune infiltration cells density levels between primary lung tumors and paired brain metastatic tumors by mIF. The box
plots display that proportions of CD3þ T cell (paired Wilcoxon ranked sum test p ¼ 0.012), CD4þ T cells (paired Wilcoxon
ranked sum test p ¼ 0.0039), and CD8þ T cells (paired Wilcoxon ranked sum test p ¼ 0.0078) are significantly lower in BMs
than in LPs. No significant differences were found in PD-L1þ cell density between LPs and BMs (paired Wilcoxon ranked sum
test p ¼ 0.65), as measured by QIF. (E) Representative mIF images revealing the simultaneous staining of panel 1 (left): DAPI
(blue), CD8þ (red), PD-1 (orange), PD-L1þ (yellow), CD68þ (pink), CD163þ (green), and CK (tumor cell, cyan); panel 2
(right): DAPI (blue), CD3þ (red), CD20þ (orange), Foxp3þ (yellow), CD56þ (pink), CD4þ (green), and CK (cyan) in primary
lung tumors and brain metastatic tumors; CD3þ T cells, CD4þ T cells, and CD8þ cells within LP have higher density levels
compared with primary lung tumors, as measured by QIF. (F) Relation of the immune infiltration score and ITH score. Left:
The pairwise ITH distances and immune distances between every paired LP and BM from the same patient (Spearman’s
correlation analysis, p ¼ 2.1E-02, R ¼ 0.52, n ¼ 22). Right: Negative correlation between the immune cell infiltration score
and ITH score in LPs and BMs (Pearson’s correlation analysis p ¼ 2.3E-06, R ¼ 0.64). (G) Negative correlation between the
CD8þ T cell density levels (mIF) and ITH score. (Pearson’s correlation analysis p ¼ 0.0396, R ¼ 0.43). BM, brain metastasis;
ITH, intratumor heterogeneity; LP, primary lung adenocarcinoma; mIF, multiplex immunofluorescence; PD-1, programmed
cell death protein 1; PD-L1, programmed death-ligand 1; QIF, quantitative immunofluorescence; ssGSEA, single-sample gene
set enrichment analysis; TIME, tumor immune microenvironment.
12 Wang et al Journal of Thoracic Oncology Vol. - No. -

A B C

D E F

Figure 5. Neoantigen depletion and immune editing in patients with LUAD with brain metastasis. (A) Top: The number of
neoantigens in a region with copy number loss is found per BM. Middle: The proportion of neoantigens lost by means of copy
number loss events in each BM and LP sample. Bottom: The number of neoantigens on a region of copy number loss is found
per LP. (B) Neoantigen gene expression levels (log2(TPM þ 1)) were found lower in CN loss regions than no CN loss (t test, p ¼
0.01). (C) The change in the expected neoantigen burden (left, red), the observed neoantigen burden (middle, yellow), and
the ratio of observed/expected neoantigen burden (right, blue) from LPs to BMs are found. The paired Wilcoxon ranked sum
test was used. (D) Correlation between the ratio of observed/expected neoantigen burden and ITH score in LPs and BMs.
There was a negative correlation between the ITH score and the observed-to-expected ratio of neoantigens (Spearman’s
correlation p ¼ 1.56E-04, R ¼ 0.54). (E) Correlation between the ratio of observed/expected neoantigen burden and
immune score in LPs and BMs. There was a positive correlation between the immune score and the observed-to-expected
ratio of neoantigens (Spearman’s correlation p ¼ 0.049, R ¼ 0.3). (F) Correlation between the ratio of observed/ ex-
pected neoantigen burden and CD4þ and CD8þ T cell density in LPs and BMs. There was a positive correlation between CD4þ
and CD8þ T cell density and ratio of observed/expected neoantigen burden (Spearman’s correlation p ¼ 6.51E-03, R ¼ 0.63).
BM, brain metastasis; CN, copy number; ITH, intratumor heterogeneity; LP, primary lung adenocarcinoma; LUAD, lung
adenocarcinoma; TPM, transcripts per million.

by the immune system, including limited antigenicity
downstream of repressed antigen expression, defects in
the molecular machinery for antigen presentation, HLA
molecule alterations or inhibitory immune checkpoint
molecules and their ligands, and anti-inflammatory cyto-
kine secretion.7,8,52–54 Nevertheless, it remains unknown
how cancer cells evade the immune system in the brain.
HLA-LOH was reported as an immune escape mechanism
to strong selection pressures.53 Unfortunately, we did not
find a difference in HLA-LOH (LOH of human leukocyte
antigen class I) events between LPs and BMs (Fisher’s
exact test, p > 0.05; Supplementary Fig. 9B) and high-
and low-ITH BMs (Fisher’s exact test, p > 0.05;
Supplementary Fig. 9C) suggesting alternative mechanism
in play underlying decreased expression of these genes.
To display the panorama of immune evasion in
brain metastasis, we mapped immune-related tumor
features according to the genetic ITH level (Fig. 6A).
Comparing high (higher than 50th percentile; n ¼ 11)
with low (lower than 50th percentile; n ¼ 11) ITH,
high-ITH BMs had a higher SCNA burden (Wilcoxon
ranked sum test p < 0.001), higher HRD score (Wil-
coxon ranked sum test p < 0.05), and lower immune
score (Wilcoxon ranked sum test p < 0.01) than the
--- 2023 Intratumor Heterogeneity of Brain Metastasis 13

B C D

Figure 6. ITH and immune escape in patients with LUAD with brain metastasis. (A) Genomic and immune characterizations for
each BM sample: ITH score, SCNA burden, HRD score, immune cell infiltrates, expression density of neoantigen gene, and
expression of genes, including HLA phenotyping-, checkpoint-, cytolytic activity-, antigen presentation-, and metabolic-related
genes. Patients were split according to the ITH score [high-ITH group (upper 50%) n ¼ 11 versus low ITH group n ¼ 11]. Red, high
values; blue, lower values. Statistics of ITH score, SCNA burden, HRD score, and immune cell infiltrates on the basis of the
14 Wang et al Journal of Thoracic Oncology Vol. - No. -

low-ITH BMs (Fig. 6A). To further explore the mecha-
nism of antigen presentation defects in high-ITH BMs,
we compared the expression of HLA genes between the
two groups and found that the expression of MHC class
I type genes, including HLA-A, HLA-B, HLA-C, and B2M,
was significantly lower (t test p < 0.05) in high-ITH
BMs (Fig. 6A). Moreover, the HLA-A/B/C and B2M
values were significantly negatively correlated with the
ITH scores in BMs (Supplementary Fig. 10, Pearson’s
correlation p < 0.05). Antigen presentation-related
genes were also differentially expressed (t test p <
0.05) between the two groups, with STAT1, STAT2, and
TAP-1 significantly down-regulated in ITH-high BMs
(Fig. 6A). Co-stimulator–related genes were also
differentially expressed (t test p < 0.05) between the
two groups, with B7-1, B7-2, CD40, ICAM-1, and LFA-1
significantly down-regulated in high-ITH BMs (Fig. 6A
and Supplementary Fig. 11A).
To provide mechanistic insights into immune escape,
we performed gene set enrichment analysis on the DEGs
from the low- and high-ITH BM groups, which revealed
that two antigen presentation pathways—the cytosolic
DNA sensing pathway (q-value ¼ 1.5E-02, enrichment
score [ES] ¼ 1.37; Fig. 6C) and the endocytosis
pathway—have significantly lower activities in ITH-high
BMs (q-value ¼ 6.6E-03, ES ¼ 1.35; Fig. 6C). In con-
trary, the cell cycle and DNA replication pathways have
significantly higher activities in ITH-high BMs (Fig. 6D).
Moreover, the NF-kappa B signaling and TGF-b signaling
pathways are two other pathways involved in antigen
processing/presentation, and CNA losses might lead to
neoantigen presentation defects. At the DNA level, we
found that approximately 73% (eight of 11 samples) of
high-ITH BMs carried CNA losses in these two pathways,
compared with only approximately 9% (one of 11 sam-
ples) of low-ITH BMs (Fisher’s exact test p ¼ 7.5E-03,
Supplementary Table 15).
Furthermore, significantly lower neoantigen expres-
sion among high-ITH BMs than among low ITH BMs was
observed (Fig. 6A; t test p ¼ 2.1E-08), suggesting a more
efficient selection of neoantigen-depletion clones within
high-ITH BMs in temporal evolution, which serve as a
main driver for immune escape.
We also found that high-ITH BMs had significantly (t
test p < 0.05) higher expression levels of metabolism-
related genes, including CYP2A7, CYP8B1, TM7SF2, and
TYRP1, indicating an increased rate of cell metabolism in
this group (Fig. 6A). Meanwhile, compared with lowest-ITH
BMs (lower 25%, n ¼ 6), another two pathways, the cell
cycle pathway and DNA repair pathway involved in cell
proliferation, were up-regulated in highest-ITH BMs (upper
25%, n ¼ 6, Supplementary Fig. 11B). These results imply
that in addition to evading the immune system, higher-ITH
BMs tend to acquire a higher growth rate.
Intriguingly, for the low-ITH BM group, we found that
inhibitory immune checkpoint molecules, such as PD-L1
and CTLA-4, were up-regulated (t test p < 0.05; Fig. 6A),
which was consistent with the mIF results (Fig. 6B). In
line with the results, PD-L1þ cell densities from mIF
were negatively correlated with ITH score in BMs
(Spearman’s correlation p ¼ 0.018, R ¼ 0.69,
Supplementary Fig. 11C). PD-L1 and CTLA-4 are immune
inhibitory receptors, the high expression of which may
reflect a cancer adaptive immune response to an active
immune system.55,56 This result suggested that unlike
high-ITH BMs, low-ITH BMs tended to escape immune
attack by up-regulation of immune checkpoints.
Taken together, these findings propose a compre-
hensive model of immune invasion mechanisms in
BMs: high-ITH BMs could develop defects in antigen
presentation that block T cell recognition though the
repression of neoantigen transcripts, the down-
regulation of HLA, and antigen presentation pathway
disorders, whereas ITH-low BMs could block T cell
recognition by inhibiting immune checkpoint molecules
and their ligands (Fig. 7).
ITH Is Prognostic in LUAD Brain Metastases
The above-mentioned results clearly reveal that ITH
plays a key role in BMs. Previous observations have
revealed that patients with low ITH in primary tumors
had significantly better survival.40,57,58 Nevertheless, the
evidence of ITH in metastases is still fragmentary and
unelucidated. Therefore, we evaluated whether stratifi-
cation of patients with LUAD brain metastasis using the

Wilcoxon ranked sum test. A t test was used for the other parts. Significant correlations are marked with *p < 0.05, **p < 0.01,
and ***p < 0.001. (B) Representative mIF images stained for PD-L1 (yellow) and DAPI (blue) on BM tissues with low (P07 and P18)
or high (P10 and P02) ITH score. Scale bar, 50 mm. (C) GSEA results of differentially expressed genes in the comparison of high-
ITH (upper 50%, n ¼ 11) versus low-ITH (lower 50%, n ¼ 11) BMs: Top and bottom reveal that the cytosolic DNA sensing pathway,
the endocytosis pathway and antigen processing and presentation were significantly down-regulated in high-ITH BMs, with a q-
value of less than 0.05 and an ES lower than 0.5. (D) GSEA results of differentially expressed genes in the comparison of
highest-ITH (upper 25%, n ¼ 6) versus lowest-ITH (lower 25%, n ¼ 6) BMs: Top and bottom reveal that the cell cycle pathway and
the DNA replication pathway were significantly up-regulated in highest-ITH BMs, with a q-value of less than 0.05 and an ES
greater than 0.5. BM, brain metastasis; ES, enrichment score; GSEA, gene set enrichment analysis; HRD, homologous recom-
bination deficiency; ITH, intratumor heterogeneity; LUAD, lung adenocarcinoma; mIF, multiplex immunofluorescence; PD-L1,
programmed death-ligand 1; SCNA, somatic copy number alteration.
--- 2023 Intratumor Heterogeneity of Brain Metastasis 15

Figure 7. Model of proposed immune escape mechanisms in LUAD brain metastasis. The model illustrates different immune
escape mechanisms in high-ITH and low-ITH BMs. During the evolutionary process of brain metastasis, the accumulation of
neoantigens may induce local immune infiltrates, leading to the killing of tumor cells. BMs with high ITH may present antigen-
presenting machinery disruption, including depletion of neoantigens, antigen presentation defects, and down-regulation of
neoantigen expression, leading to low immune cell infiltration, including decreased CD8þ T cells and DC cells. Alternatively,
during the evolution process, subclones with low ITH levels may induce up-regulation of the PD-1/PD-L1 axis, which may be
another mechanism of immune escape. Other subclones may evade killing through other mechanisms. BM, brain metastasis;
DC, dendritic cell; ITH, intratumor heterogeneity; LUAD, lung adenocarcinoma; PD-1, programmed cell death protein 1; PD-
L1, programmed death-ligand 1.

ITH score could provide predictive power in prognosis.
As expected, patients with low-ITH (lower 50%) brain
metastasis had significantly longer overall survival (log-
rank p ¼ 4.2E-03; Fig. 8A) than high-ITH patients.
This observation remained significant in a multivariate
model incorporating age, sex, immune infiltration score,
smoking, stage, and TMB as variables (p < 0.05;
Supplementary Fig. 12A). Furthermore, we observed that
patients with either a high ITH or a high ITH ratio (upper
50% in BMs) in BMs had significantly decreased overall
survival (log-rank p ¼ 1.5E-02; Fig. 8B).
According to the ITH score of LPs, we also grouped
patients into two groups (high versus low ITH), defined
as the upper quartile cohort versus the rest. The high-
ITH LP group had worse disease-free survival but, un-
like the high-ITH BM group, it was not associated with
overall survival (log-rank p ¼ 0.045; Supplementary
Fig. 12B and C) (Methods). These results suggest that it
is important to consider ITH when predicting clinical
outcomes and that the role of ITH in LP and BM prog-
nosis could be different.
The relationships between ITH dynamics and
prognosis were further illustrated in patients with
follow-up and treatment (Fig. 8C and D). Patient P15,
a 58-year-old woman without a history of smoking,
was characterized as having low risk by the ITH
score. She underwent lobectomy for LUAD in October
2011, was staged as pT1bN0M0, and was radiologi-
cally diagnosed with brain metastasis after regular
follow-up. She received EGFR tyrosine kinase inhibi-
tor treatment, as sequencing analysis revealed that
the tumor had an EGFR 19 deletion mutation. Her
disease has been stable thus far, and she has a
promising survival time (more than 9 y after lobec-
tomy and 3 y after BM resection). In contrast, pa-
tients characterized as having high risk by the ITH
score have a different story. Patient P02 was a 58-
year-old female nonsmoker who underwent lobec-
tomy for LUAD in October 2011 and was staged as
pT2aN0M0. Owing to solitary brain metastasis, she
survived only 22 months after lobectomy. Patient
P12 had an EGFR L858R mutation and was treated
with EGFR tyrosine kinase inhibitor. Her disease
progressed quickly, and she did not survive after 16
months. Thus, our prediction model stratified pa-
tients with BM into low- and high-risk groups, which
were confirmed to have significantly different sur-
vival outcomes.
16 Wang et al Journal of Thoracic Oncology Vol. - No. -

A B

C D

Figure 8. Prognostic impact of intratumor heterogeneity on patients with LUAD with brain metastasis. (A) Kaplan-Meier curve
for the overall survival of patients with BM with high ITH (defined as BM ITH score top 50%) and low ITH (defined as BM ITH
score bottom 50%). (B) Kaplan-Meier curve that combines the BMs ITH score and the LPs ITH score (the high-risk group is
defined as BMs ITH score top 50% and BM ITH score/paired LP ITH score greater than median (BM ITH score/paired LP ITH
score); alternatively, the low-risk group is defined as BMs ITH score bottom 50% and BMs ITH score/LPs ITH score less than or
equal to median BM ITH score/paired LP ITH score). (C, D) Timelines with clinical and treatment data from the time of
diagnosis to the last follow-up or time of death for four patients in our study; (C) reveals patients P15 and P05 in the low-risk
group with longer survival; (D) reveals patients P02 and P12 in the high-risk group with poor prognosis. Each sample was
labeled and annotated with a colored rectangle for the first surgery, adjuvant therapy, therapy after brain metastasis, time
lag, and image from the primary lung tumors and brain metastasis resection. The time intervals between surgical inter-
vention and death (last follow-up) (represented by various line widths) and the treatment information are found. The sites of
metastasis are colored and represented at the body location. BM, brain metastasis; ITH, intratumor heterogeneity; LP,
primary lung adenocarcinoma; LUAD, lung adenocarcinoma.

Discussion lung cancer, survival related to metastatic cancer, espe-

Metastasis is believed to be an evolutionary process cially brain metastasis, remains poor.3 Herein, we
shaped by the dynamic interaction of tumor cells with established a brain metastasis cohort with a relatively
host antitumor immune surveillance.59 Despite remark- large number of paired samples with detailed clinical
able progress in understanding and treating primary information and a longer follow-up time. To capture the
--- 2023
complex interplay between cancer genomic alterations
and antitumor immunity in patients with BM with LUAD,
we integrated genomic heterogeneity, immune infiltra-
tion, and prognostic information to construct a dynamic
evolutionary model revealing how tumors are shaped by
the immune microenvironment and to investigate the
mechanisms of immune escape during tumor metastasis.
Comparisons between primary and brain metastases and
within brain metastases revealed several novel findings
as follows on the power of intratumor heterogeneity
attributed to intrinsic (genomic instability) and extrinsic
(immunosurveillance) selection in brain metastasis
progression and development.
First, by delineating the tumor mutational landscape
and its evolutionary trajectory, we found a widespread
genetic ITH increase in the temporal dimension (from
LPs and BMs). BMs exhibited a significantly higher ITH
level than LPs, which was mainly caused by HRD alter-
ations or other SCNA-associated mechanisms, repre-
senting increased genomic instability and genomic
complexity in BMs. We found that an increase in genetic
ITH levels, considered a substitute for subclonal di-
versity,35,41,60,61 is a result of ongoing tumor evolution
and clonal selection. Importantly, ITH has emerged as
the mechanism of tumor escape from immuno-
surveillance, progression, and resistance to immuno-
therapy.10,35,41,62–64 In our settings, the temporal
dynamics of ITH were first and comprehensively eluci-
dated in BMs from LUAD. The enforcement of HRD in the
development of BM suggested that HRD could be a late
event and evolves independently from primary lung
cancer, accumulating an aging-related signature. Of note,
our results revealed that a high degree of ITH in BMs, not
LPs, represents a negative prognostic factor for patients.
The correlation of ITH with clinical outcome reveals the
significance of ITH temporal dynamics in tumor metas-
tasis evolution. Thus, the genetic ITH elevation in BM
offers a fertile soil for tumor evolution and metastasis,
ultimately shaping genomic features and the immuno-
genicity of the tumor.
Second, our parallel model suggested that the tem-
poral evolution of ITH from LPs to BMs is determined by
the dynamic nature of its sources, which include not only
tumor-intrinsic processes such as genetic instability but
also features of the TIME. Temporal immune microen-
vironment remodeling in brain metastasis results in cold
immunologic profiles. Such changes encompass the
depletion of CD8þ T cells and DCs coupled with the
accumulation of immunosuppressive cells such as
myeloid-derived suppressor cells. M2 macrophages, Treg
cells, and decreased effector functions were observed in
BMs but not in LPs. Moreover, we found that the tem-
poral alteration in ITH has a negative impact on tumor
neoantigens (neoantigen depletion), which are important
Intratumor Heterogeneity of Brain Metastasis 17
targets for tumor-specific CD8þ cytotoxic T lympho-
cytes. This finding supported our hypothesis that in
metastasis evolution, neoantigen-depleted subclones
existing in the presence of ITH were enriched by im-
mune pressure. This mechanism of immunoediting was
first reported through neoantigen loss accompanied by
ITH dynamics in the evolutionary process of brain
metastasis, which has significant implications for
immunotherapy strategies.
Third, it has been reported that metastatic tumor
cells in different target organ microenvironments
require different adaptive mechanisms to successfully
colonize and proliferate.12 Importantly, we observed that
high or low ITH in BM under immune selection pressure
would drive the tumors to adapt distinct immune escape
mechanisms in brain metastasis. Our results, to the best
of our knowledge, are the first to reveal evidence of
brain metastasis evolving through different immunoe-
diting mechanisms stratified by ITH levels, either antigen
presentation disruption (high-ITH group) or immune
checkpoint gene down-regulation, such as PD-L1/CTLA-
4 (low-ITH group), at both the DNA and RNA levels. In
detail, high-ITH BMs had neoantigen presentation
disruption by suppressing antigen expression or muta-
tion defects in the neoantigen presentation pathway,
including HLA genes, STAT1, STAT2, TAP-1, the cytosolic
DNA sensing pathway, the endocytosis pathway, the NF-
kappa B signaling pathway, and the TGF-b signaling
pathway. For BMs with low levels of ITH and high levels
of immune infiltration, however, immune escape tends to
be achieved through mechanisms including up-regulated
checkpoint molecule expression and the reinforcement
of immunoregulatory cell recruitment (Fig. 7). It is sug-
gested that high-ITH BMs tend to evade the immune
system by causing defects in antigen presentation
through many mechanisms, including suppressing the
expression of neoantigens, MHC class I type genes, and
other antigen presentation-related genes and increasing
CNA losses in presentation-related pathways. In addi-
tion, we discovered that an elevation in ITH accompa-
nied by a TME characterized by limited immune
infiltration in BM samples seems to correlate with poor
prognosis. Given previous findings, we proposed that
because tumor cells with high ITH are inherently
genetically unstable, tumor cells in equilibrium under
strong immune pressure may facilitate immune evasion
and progression through antigen presentation process
disruption.
Last, the overwhelming intratumor heterogeneity at
the genetic level has a major impact on the efficacy of
various therapies, in particular immunotherapy, sug-
gesting that the biopsy of intracranial lesions is much
more informative than that of the primary tumor to
guide therapy selection for the successful treatment of
18 Wang et al
brain metastases. PD-L1 was regarded as a predictor
for immune checkpoint inhibitor (ICI) therapy; how-
ever, some patients with negative PD-L1 expression
also achieved clinical benefits from ICI treatment. PD-
L1 expression varied among different biopsy sites in
NSCLC, with high levels in liver and adrenal metasta-
ses and low levels in brain and bone metastases,
whereas no significant change was reported between
brain metastasis and lung biopsies.65 We did not
observe significant change of PD-L1þ cells between
LPs and BMs in our work either (Fig. 4D). Interest-
ingly, PD-L1 expression was found to be negatively
correlated with ITH score in BMs. ITH was revealed as a
potential biomarker that can predict the efficacy of ICI
therapy in patients with advanced NSCLC and other
tumors.66 Temporal ITH may emerge and drive aggres-
sive disease progression in the context of resistance to
therapy of BMs. Thus, targeting a common mechanism
that is least likely to be influenced by tumor genomic
heterogeneity may be a viable strategy. Nevertheless,
comparative analyses of the TIME of brain metastases
will likely yield novel insights into immunotherapies for
patients with BM by targeting recruiting immunosup-
pressive cells, including myeloid-derived suppressor
cells and Treg cells, and their immunosuppressive pro-
files/properties. Taken together, our findings suggest
that combining therapies targeting different tumor
mechanisms of immune evasion (anti-programmed cell
death protein 1/PD-L1 in low-ITH patients and PARP
inhibitors in high-ITH patients) may provide optimal
benefits for patients with BM. We also generated a
prognostic predictive model for patients with BM and
provided patient stratification criteria for therapeutic
interventions.
In our study, we revealed evidence that tumor
heterogeneity sculpts clonal evolution to go different
directions under the pressure of the immune system.
Our tumor immunity model of LUAD-BM proposed
that the development of tumor clones is linked to the
intrametastatic immune microenvironment by means
of the immunoediting process. Future studies could
use single-cell RNA-seq and immune profiling to study
the landscape of genotype–immune phenotype in-
teractions in larger cohorts of patients with brain
metastatic NSCLC. Understanding the molecular
evolutionary processes of lung cancer and BM may
offer greater insight into the development of better
and more precise treatments. Our elucidation of the
major sources of ITH and the key temporal features of
ITH dynamics under immune pressure is paramount
for the development of effective treatments for brain
metastasis.
In summary, we highlighted the complex in-
teractions between tumor cells and the tumor-
Journal of Thoracic Oncology Vol. - No. -
associated microenvironment and discussed the mech-
anism of immune escape between tumor cells and type-
specific cells in brain metastatic microenvironments.
The extensive multiomic profiling of paired LUAD and
brain metastasis tissues analyzed here has provided
unique highlights on how metastases develop and
evolve, how genetic and TIME features vary, how their
predicted neoantigen landscapes are altered, and how
ITH varies by TIME reshaping. The better and more
precise understanding of the metastatic process and
escape mechanism from cancer immunoediting ob-
tained in our study may open the door for the devel-
opment of novel therapeutic options and pave the way
for precise immunotherapeutic strategies for patients
with brain metastasis.
CRediT Authorship Contribution
Statement
Xin Wang: Methodology, Formal analysis, Data
curation, Investigation, Project administration, Writing—
original draft, Writing—review and editing.
Hua Bai: Methodology, Formal analysis, Data cura-
tion, Investigation, Resources, Writing—original draft,
Writing—review and editing.
Jiyang Zhang: Methodology, Formal analysis, Data
curation, Investigation, Software, Writing—original draft,
Writing—review and editing.
Zhijie Wang: Formal analysis, Data curation, Inves-
tigation, Resources.
Jianchuan Duan: Formal analysis, Data curation,
Investigation, Resources.
Hongqing Cai: Formal analysis, Investigation,
Resources.
Zheng Cao: Formal analysis, Data curation,
Investigation.
Qingtang Lin: Formal analysis, Investigation,
Resources.
Xiaosheng Ding: Formal analysis, Investigation,
Resources.
Yiting Sun: Investigation, Data curation.
Wei Zhang: Writing—review and editing,
Visualization.
Xiaoya Xu: Investigation, Validation.
Hao Chen: Methodology, Investigation, Software.
Dadong Zhang: Investigation, Formal analysis.
Xiaoli Feng: Data curation, Formal analysis.
Jinghai Wan: Data curation, Investigation.
Jianjun Zhang: Supervision.
Jie Wang: Conceptualization, Funding acquisition,
Project administration, Resources, Supervision, Writing—
original draft, Writing—review and editing.
Jie He: Conceptualization, Funding acquisition, Proj-
ect administration, Resources, Supervision, Writing—
original draft, Writing—review and editing.
--- 2023
Acknowledgments
This work was supported by the National Key Research and
Development Project to Dr. Jie Wang (2019YFC1315700)
and Dr. Jie He (2020AAA0109500); the NSFC Key Program
to Dr. Jie Wang (81630071); and the Beijing Municipal
Science & Technology Commission to Dr. Jie He
(Z191100006619117).
The authors thank the support and participation of
the pathologists and patients in this study.
Ethics Approval and Consent to
Participate
This study protocol was reviewed and approved by the
Institutional Ethic Committee in the Cancer Hospital, Chi-
nese Academy of Medical Sciences (NCC2016G-074). All
patients were consented about human specimen collection.
Availability of Data and Materials
The somatic mutation and tumor immune infiltration of
The Cancer Genome Atlas LUAD can be obtained from
GDC data portal (https://gdc.cancer.gov/about-data/
publications/panimmune, https://xenabrowser.net/).
The sequencing data have been deposited in the National
Genomics Data Center (https://ngdc.cncb.ac.cn/bioproject/)
under the accession number PRJNA018926. All other
data are available from the correspondence author on
reasonable request.
Supplementary Data
Note: To access the supplementary material accompa-
nying this article, visit the online version of the Journal of
Thoracic Oncology at www.jto.org and at https://doi.
org/10.1016/j.jtho.2023.09.276.
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