Biomarkers in Precision Cancer Immunotherapy

DEVELOPMENTAL THERAPEUTICS—IMMUNOTHERAPY
Biomarkers in Precision Cancer Immunotherapy:

Promise and Challenges
William B. McKean, MD, PhD1; Justin C. Moser, MD2; David Rimm, MD, PhD3; and Siwen Hu-Lieskovan, MD, PhD1
overview
The rapid expansion of modern cancer immunotherapeutics has led to a dramatic improvement in patient
survival and sustained remission for otherwise refractory malignancies. However, a significant limitation
behind these current treatment modalities is an irregularity in clinical response, which is especially pro-
nounced among checkpoint inhibition. This unpredictability leads to significant side effects, financial costs,
and health care burden, with unsatisfactory clinical benefit in the majority of treated patients. Additionally,
although ongoing studies and trials investigate the use of multiple biomarkers predictive of patient response or
harm, none of these are comprehensive in predicting potential benefit. This unmet need for validated bio-
markers is largely secondary to a prohibitive complexity within tumor parenchyma and microenvironment,
dynamic clonal and proteomic changes to therapy, heterogenous host immune defects, and varied stan-
dardization among sample preparation and reporting. Herein, we discuss current advantages of predictive
biomarkers, as well as limitations in their clinical use and application. We also review future directions, ideal
characteristics, and trial design needed for proper precision immuno-oncology and biomarker development.
INTRODUCTION Adoptive cell therapies have also shown sustained

Immuno-oncology has witnessed a revolution of basic treatment effects among otherwise refractory and
science advances and novel clinical therapies over the heavily pretreated patients. Since 2017, FDA approval
past decade. Advanced malignancies, such as stage IV of tisagenlecleucel for B-cell precursor ALL and axi-
melanoma, lung cancer, and relapsed/refractory acute cabtagene ciloleucel for diffuse large B-cell lymphoma
lymphoblastic leukemia (ALL), have shown dramatic has demonstrated impressive CR rates (approximately
improvements in overall survival (OS), progression-free 90% and 50%, respectively).3 In many cases, there is
survival (PFS), and complete response (CR) with the also evidence of chimeric transgene sequences per-
introduction of therapies such as immune checkpoint sisting within peripheral blood for years after initial
inhibition (ICI), bispecific antibodies, and chimeric treatment, suggestive of a more permanent cancer
antigen receptor (CAR) T cells. “immune surveillance.”4,5 Such promising features of
CAR T-cell therapy have inspired the rapid expansion
Anti–CTLA-4 therapy was originally approved by the of clinical studies in solid and hematologic malig-
U.S. Food and Drug Administration (FDA) in 2011 for nancies alike, with a global increase to 364 active trials
advanced melanoma following multiple clinical ob- by early 2019.6
servations that it prolonged OS.1 Since that time, the
repertoire of checkpoint inhibitors and their onco- Despite these successes, the majority of patients who
logic targets have expanded dramatically. With the qualify for immune therapy derive no benefit, as a re-
incorporation of PD-1 and PD-L1 targets, six additional sult of primary or secondary resistance to therapy.
antibodies have been approved for use against a variety Such treatment failures occur even in ICI patient co-
Author affiliations of malignancies, from cervical cancer to advanced horts, despite respective prescreening with biomarkers
and support gastroesophageal carcinomas. Recent estimates sug- such as PD-L1 tumor proportion scores or combined
information (if
gest that patient candidacy for ICI has increased from positive scores. Other predictive models such as tumor
applicable) appear mutation burden (TMB), although validated in several
at the end of this 1.54% in 2011 to 43.63% in 2018, and total re-
sponders have increased from less than 0.14% to clinical trials,7-10 show limitations under a variety of
article.
12.46%.2 An especially attractive feature of ICI has circumstances, including prior immunotherapy,11-14
Accepted on April
24, 2020 and been a durable response in subsets of patients, evi- mutational variation (including copy number alter-
published at denced by a plateau of the treatment “tails” in Kaplan- ations and indels),15 or concomitant chemotherapy.
ascopubs.org on
XXXX, XX: DOI https://
Meier survivorship curves. Rather than ultimately Importantly, although more predictive models of ICI
doi.org/10.1200/ descending to baseline, these curves flatten before the response have recently been identified, broader ap-
EDBK_280571 end of surveillance. plication is incomplete. Meta-analysis and systematic
2020 ASCO EDUCATIONAL BOOK | asco.org/edbook e275
Downloaded from ascopubs.org by 49.130.130.133 on May 27, 2020 from 049.130.130.133

Copyright © 2020 American Society of Clinical Oncology. All rights reserved.
McKean et al
downregulate priming and activity of cytotoxic responses.

Following initial cloning and characterization of the key
PRACTICAL APPLICATIONS
components,80 the PD-1/PD-L1 checkpoint was specifically
• Understanding advances in current biomarker shown to be manipulated by malignant cells to avoid host
design helps to guide appropriate application of
immunity.81 Concurrently, data were published reporting
immunotherapy.
that anti–CTLA-4 therapy promotes tumor shrinkage and
• Tumoral complexity, clonal evolution, and secondary immunity in preclinical models of sarcoma and
heterogenous host immune defects limit the colorectal adenocarcinoma.82 ICI has since become the
utility and predictability of standard biomarker
most prolific approved cancer immunotherapy, highlighted
options.
by Dr. Tasuku Honjo and Dr. James Allison receiving the
• Ideal biomarker design will require a multifac- Nobel Prize in Physiology or Medicine 2018 for their pio-
eted and largely individualized design to guide neering contributions to the field.
therapy selection and duration.
PD-L1
• Further validation of novel biomarkers through
adaptive clinical trials will require more com- PD-L1 is the only biomarker validated for its predictive utility
prehensive data collection before and during in prospective clinical trials and approved by the FDA as
treatment. a companion diagnostic prior to ICI therapy. Anti–PD-1 was
approved in metastatic non–small cell lung cancer (NSCLC)
review show similar correlation between patient response to as first-line monotherapy for patients with partial or com-
PD-1/PD-L1 therapy and TMB, PD-L1 immunohistochem- plete surface PD-L1 staining (tumor proportion scores) on
istry (IHC), or gene expression profiles (GEPs).16 However, 1% or more of malignant cells.19 Related immunohisto-
improved sensitivity, specificity, positive predictive value, chemical tests have been developed that measure PD-L1+
and positive likelihood ratio are seen with multiplex IHC tumor and immune cells relative to total tumor population
(mIHC)/immunofluorescence (IF). Such advances require (combined positive score). This assay is required for
further clinical use and development. anti–PD-1 approval in a variety of other indications, in-
cluding first-line treatment for advanced head and neck
Another complication of modern immunotherapy is the squamous cell carcinoma (HNSCC),20 second-line treat-
development of unpredictable and unique toxicities. Immune- ment for esophageal squamous cell carcinoma,21,22 third-
related adverse events (irAEs), which are considered sec- line treatment for recurrent gastric/esophagogastric junction
ondary to off-target inflammation induced by ICI therapy, can adenocarcinoma,23 second-line treatment for progressive
have a widely variable onset and affect a diversity of tissues or cervical cancer,24 and bacillus Calmette-Guérin–refractory
organ systems. Similarly, patients receiving CAR T-cell therapy high-risk nonmuscle invasive bladder cancer. These and
must be closely monitored for symptoms related to immune other studies9,25-28 highlight the versatility of PD-L1 as
effector cell–associated neurotoxicity syndrome or cytokine a positive prognostic correlate for OS across a wide spec-
release syndrome (CRS). trum of malignancies.
Discrepancies in patient treatment responses and devel- Despite their broad utility, programmed death ligand levels
opment of unpredictable toxicities highlight a special need remain an incomplete test for universal prescription of
for predictive biomarkers in immuno-oncology to further checkpoint inhibitors. Multiple studies have shown an ab-
select appropriate patient treatment cohorts. 17,18 Un- sence of association between PD-L1 expression and OS in
fortunately, the current spectrum for such biomarkers is ICI therapy.7,13,83,84 Still other cohorts have patients with
extremely limited, especially those established in large minimal or absent PD-L1 tumor expression that derive
prospective clinical trials. Additionally, substantial variation sustained responses to checkpoint inhibition. A recent
between individual patient tumors, their microenvironment, retrospective analysis of clinical trials (2011–2019) prompting
and treatment history highlights the need for more per- FDA approval of checkpoint inhibitor regimens identified
sonalized diagnostics and therapy. This review describes PD-L1 as a predictive biomarker in only 28.9% of cases.85
recent advances and established biomarkers in cancer This is attributable, in part, to dynamic host immunity; rapid
immunotherapy (Table 1) while exploring limitations behind induction of PD-L1 levels following adaptive responses to
current predictive tests and models. We propose potential therapy may not be representative of baseline patient test-
trial design and ideal biomarker use to overcome these ing. Other potential explanations for these discrepancies are
limitations in search of more precision medicine. discussed in later sections.
IMMUNE CHECKPOINT INHIBITION BIOMARKERS Tumor Mutation Burden and Microsatellite Instability
Early discoveries in the 1990s demonstrated that specific The number of genetic alterations within a tumor genome is
T-cell protein receptors—PD-1 and CTLA-4—function to considered correlative with mutant protein burden and,
e276 2020 ASCO EDUCATIONAL BOOK | asco.org/edbook

Biomarkers in Precision Cancer Immunotherapy
TABLE 1. Characteristics and Clinical Correlates of Biomarkers in Cancer Immunotherapy

Biomarker Immunotherapy Malignancy Example Tests Clinical Utility Tissue Source
PD-L1 Checkpoint inhibition Multiple TPS,19 CPS,20–24 IC expression25 CR/PR26 Tumor, infiltrating
21,23–25,27,28 lymphocytes/
ORR
macrophages
DCB/DOR21,23
PFS28
OS19,20,28
TMB Checkpoint inhibition Multiple ΔTMB 12
CR/PR13,25,29 Tumor
9,30–32
ORR
DCB/
DOR30,33,34
PFS9,30,31,34
OS12,13,31,34
MMR/MSI Checkpoint inhibition Multiple N/A ORR35 Tumor
35
PFS
OS35
36
Aneuploidy Checkpoint inhibition Multiple SCNA level CR/PR37 Tumor
36
OS
TIL Checkpoint inhibition Multiple Immunoscore,38–40 CTLA-4hiPD-1hi,41 CR/ Infiltrating
TRM,42,43 PD-1T,44 4PD1hi,45 PR12,41,44,47,48 lymphocytes
CYT12,46
ORR49,50
PFS41,50
OS42,44,48
51–55 56
GEP Checkpoint inhibition Multiple IFN-γ signature, IMPRES, CR/PR52,53,55–58 Tumor, infiltrating
TIDE,57 immunophenoscore58 54 lymphocytes
ORR
PFS53–57
OS51,54–57
mIHC/IF Checkpoint inhibition Melanoma, MCC, N/A CR/PR47,59,60 Tumor, infiltrating
NSCLC 50 lymphocytes
ORR
DCB/DOR61
PFS50,59,61
OS59,61
62 89
PET/CT imaging Checkpoint inhibition Multiple Immune-PET (i.e., ZR- CR/PR63,64 N/A
desferrioxamine), 18F-FDG62,63 64
PFS
OS64
Circulating CAR Adoptive cell therapy B-cell precursor N/A ORR65 Peripheral blood
T cells ALL, DLBCL
DFS66
Peripheral Checkpoint inhibition, Multiple Treg,67 CD27+CD45RO CD8+,68 CR/PR67,68 Peripheral blood
lymphocytes adoptive cell therapy, RLC69
OS69
T-cell engagers
(Continued on following page)

McKean et al
TABLE 1. Characteristics and Clinical Correlates of Biomarkers in Cancer Immunotherapy (Continued)

Biomarker Immunotherapy Malignancy Example Tests Clinical Utility Tissue Source
70–77
Proinflammatory Checkpoint inhibition, Melanoma, B-cell IFN-γ, IL-13, MIP-1α, IL-6, IL-8, Toxicity Peripheral blood
cytokines adoptive cell therapy precursor ALL, sCD25, IL-17, sCD163, CXCL5
CLL, NHL
Autoantibodies Checkpoint inhibition Multiple Anti-thyroglobulin, anti-GAD65, anti- Toxicity78,79 Peripheral blood
IA2, anti-insulin, anti-ZnT8
Abbreviations: TPS, tumor proportion score; CPS, combined positive score; IC, infiltrating immune cell; CR, complete response; PR, partial response; ORR,
objective or overall response rate; DCB, durable clinical benefit; DOR, duration of response; PFS, progression-free survival; OS, overall survival; TMB, tumor
mutation burden; ΔTMB, change in TMB; MMR/MSI, mismatch repair/microsatellite instable; N/A, not applicable; SCNA, somatic copy number alteration;
TIL, tumor-infiltrating lymphocyte; TRM, tissue-resident memory T cell; PD-1T, intratumoral CD8+ T cell PD-1 elevation; 4PD1hi, unconventional PD-
1–expressing CD4+FOXP3 subpopulation; CYT, cytolytic score; IMPRES, immune-predictive score; GEP, gene expression profile; IFN-γ, interferon gamma;
TIDE, tumor immune dysfunction and exclusion; mIHC, multiplex immunohistochemical; IF, immunofluorescence; MCC, Merkel cell carcinoma; NSCLC,
non–small cell lung cancer; FDG, fluorodeoxyglucose; CAR, chimeric antigen receptor; ALL, acute lymphocytic leukemia; DLBCL, diffuse large B-cell
lymphoma; DFS, disease-free survival; Treg, regulatory T cell; RLC, relative lymphocyte count; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin
lymphoma; IL-13, interleukin-13; MIP-1α, macrophage inflammatory protein 1α; IL-6, interleukin-6; IL-8, interleukin-8; sCD25, soluble interleukin-2
receptor; IL-17, interleukin 17; sCD163, soluble cluster of differentiation 163; CXCL5, CXC motif chemokine 5; GAD65, glutamic acid decarboxylase 65; IA2,
α-islet antigen 2; ZnT8, zinc transporter 8.
thus, host immunity against “non-self” neoantigens. Non- MMR deficiency independently correlates with ICI efficacy.
synonymous single nucleotide variants, collectively known Tumors that demonstrate deficiency in MMR (and, thus, are
as TMB, are the most commonly associated mutational high in MSI) have improved response to checkpoint in-
variant with ICI therapy response. Initial studies evaluating hibition. These data were first demonstrated in phase II
cohorts of patients with melanoma treated with ipilimumab29,33 studies evaluating anti–PD-1 therapy among patients with
and patients with NSCLC on pembrolizumab30 demonstrated progressive and heavily pretreated metastatic colorectal
higher levels of nonsynonymous single nucleotide variants carcinoma.35 Objective response rate (ORR) to pembrolizumab
among patients with improved clinical response and PFS. among MSI-high cohorts increased to 40% (compared with
Since these data, positive associations with checkpoint in- 0% in MSI-stable), and median PFS and OS were not
hibitor response and TMB have been demonstrated in several reached. Subsequent experiments among multiple tumor
other malignancies, including small cell lung cancer,31 uro- types (including prostate, pancreas, thyroid, neuroendo-
thelial carcinoma,25 and HPV HNSCC.13 TMB may have even crine, endometrial, gastroesophageal, and biliary) repro-
broader predictive applications across a range of tumor types. duced response and survival benefits among patients with
For example, a meta-analysis assessing objective response MMR deficiency (radiographic response, 53%; CR, 21%;
rate to ICI therapy across 27 cancer subtypes demon- median PFS and OS not reached).87 Accelerated approval for
strated positive correlation in linear regression with logarithmic pembrolizumab as second-line therapy for MMR-deficient
measurements of TMB.32 Indeed, a very high response rate to solid malignancies was granted by the FDA consequent to
anti–PD-1 therapy was seen in a cohort of patients with these findings.
desmoplastic melanoma, one of the tumor types that has the Although TMB appears promising, there are substantial
highest TMB.86 More recently, high nonsynonymous somatic concerns related to its clinical implementation. Among the
TMB assessed by next-generation sequencing correlated more important of these is the lack of assay standardization.
positively with OS among a large cohort of patients with ad- Although a number of standardization efforts have begun,
vanced or metastatic cancers (including NSCLC, melanoma, none have yet been published. Standardization is further
HNSCC, renal cell carcinoma, and bladder cancer; 1,662 complicated by different tests using different criteria. For
patients).34 This study suggests an important feature of TMB as instance, one of the more popular assays, the Foundation
a biomarker: even relative increases among histologic sub- Medicine test, uses synonymous and nonsynonymous
types with canonically low mutational burden correlate with mutations, in conflict with the original concept of neoantigen
immunotherapy response. In these data, the top 20% of so- detection (by definition, synonymous mutations do not
matic nonsynonymous single nucleotide variants relative to generate neoantigens). An equally significant issue is the
specific cancer histologies was predictive of improved OS definition of the TMB cut-point. For example, although some
within each group. groups used 10 mutations per megabase pair, others used
Mismatch repair (MMR) status is an underlying genetic different values. Perhaps the best analysis of this issue is
process that contributes to neoantigen formation and TMB. illustrated by Samstein et al34; the hazard ratio (HR) for OS
The presence of microsatellite instability (MSI) secondary to following ICI is plotted against TMB cut-points among

multiple advanced cancers. This curve shows that the HR PD-1 levels.44 Importantly, increased PD-1T CD8+ T-cell
declines as mutations per megabase pair increase, reaching levels strongly improved OS and response to nivolumab
a plateau at about 23 to 25 mutations per megabase pair. in this cohort. Similarly, among patients with metastatic
These data suggest that the cut-point for clinical testing melanoma, increased TIL expression of PD-1 and CTLA-4
should be more than 20 mutations per megabase pair; (CTLA-4hiPD-1hi) correlates with improved response and
however, the disadvantage of this threshold is that only PFS after treatment with anti–PD-1 therapy.41 Conversely,
a small percentage of any population exceeds that level preclinical studies suggest that more chronic stimulation of
of TMB. PD-1 surface expression can terminally exhaust CD8+
T cells through regulatory T-cell (Treg) recruitment or epi-
Tumor-Infiltrating Lymphocytes
genetic or transcriptional modulation,96-100 blunting their
Checkpoint inhibition unleashes cytotoxic T-cell activity response to ICI. Additionally, unconventional PD-1–expressing
through direct blockade of CTLA-4 or PD-1 checkpoint CD4+FOXP3 subpopulations are proportional to tumor bur-
signaling. Within tumoral stroma and parenchyma, this den among patients with melanoma or NSCLC and correlate
serves to reactivate otherwise exhausted effector T-cell inversely with OS among cohorts with NSCLC cohorts.45 Un-
populations made quiescent by antihost countermea- usually, CTLA-4 inhibition is shown to increase unconven-
sures. As a correlative, the density and location of T lym- tional PD-1–expressing CD4+FOXP3 subpopulation cell
phocytes within the tumor and its microenvironment could counts within tumors and peripheral blood, whereas PD-1
predict ICI response. For example, an “immune-inflamed” inhibition has the opposite effect.
signature is characterized by infiltration of CD8+ and CD4+
Other TIL immunophenotypes have been proposed as
T cells within tumoral parenchyma and in close proximity to
predictive biomarkers for ICI patient response. For example,
malignant cells.88 This phenotype is associated with im-
not all subsets of effector T cells subjected to prolonged
proved responses and OS among patients treated with
stimuli lose responsiveness to checkpoint inhibition. CD8+
checkpoint inhibition for a variety of malignancies.47,49
T cells coexpressing T cell factor 1 and PD-1, which are
Among cohorts with early-stage colorectal carcinoma,
present during chronic infections, are expanded intra-
quantification of cytotoxic and memory T-cell populations
tumorally and peripherally by checkpoint inhibition and
within tumoral cores and invasive margins (Immunoscore)
correlate positively with immunotherapy response.101-103 In
was more prognostic of survival and disease recurrence
addition, tissue-resident memory T cells—another recently
than MSI status or TNM staging.38,39,89,90 Similar tumor
identified population—have been linked with immune re-
profiling with Immunoscore is being evaluated in patients
sponse and prognosis among patients with solid tumors
with melanoma or NSCLC treated with ICI.40,91,92 In-
(e.g., breast cancer and NSCLC).42,43 Expression of integrins
terestingly, studies in melanoma suggest that the tumor-
such as CD103 and CD49a is thought to provide tissue-
infiltrating lymphocyte (TIL) density at the invasive margin
resident memory T cells with the ability to persist in non-
alone is most correlative with checkpoint inhibitor (anti–PD-
lymphoid tissues (including tumor parenchyma), whereas
1) response.47,50
increased levels of granzyme B, tumor necrosis factor-α,
By extension, tumors that lack parenchymal invasion of T interferon-γ (IFN-γ), PD-1, and CTLA-4 make them espe-
lymphocytes are resistant to ICI therapy. This can result from cially cytotoxic upon checkpoint inhibition.104,105 Even
T-cell exclusion to the surrounding stroma via WNT/β- simply measuring expression of cytolytic activity among
catenin or transforming growth factor-β signaling (“immune effector T cells may serve as an effective biomarker in ICI.
excluded”)48,88,93-95 or a complete lack of effector T-cell Among cohorts with progressive melanoma, the geometric
infiltration in the parenchyma or stroma (“immune des- mean of granzyme A and perforin 1 (cytolytic score) was
ert”). In broader terms, immune-excluded tumors, although shown to better predict response to nivolumab therapy than
sensitized to host immunity, actively evade detection with TIL density,12 and it correlates strongly with TMB.46
countermeasures that include active expression of PD-1 Importantly, although pathologic analysis of TILs can be
and CTLA-4. Alternatively, immune-desert tumors are com- predictive of response to immunotherapy, tissue biopsy is
pletely agnostic to host immune response. not possible in all patients. As a consequence, less invasive
In addition to density and intratumoral location, immu- biomarkers are needed. A significant advancement in pre-
nophenotype is a level of TIL diversity that can be in- cision medicine and biomarker classification is the use of
dependently associated with patient ICI response. For advanced imaging to characterize tumors.62-64 Of note,
instance, PD-1 expression among TILs correlates with a novel technique for detecting whole-body CD8+ T-cell
a variety of outcomes. In patients with stage IV NSCLC, subsets using 89ZR-desferrioxamine-labeled cys-diabodies
increased cell surface expression of PD-1 among intra- (immune-PET) has been validated in anti–PD-L1 preclinical
tumoral CD8+ T cells (PD-1T) disposes to improved tumor models and is currently undergoing a phase II clinical
reactivity compared with normal, absent, or virally induced trial.106

McKean et al
Gene Expression Profiles among immune-activating and immune-inhibitory states. For

Gene expression signatures is a rapidly expanding and instance, close analysis between spontaneously regressing or
comprehensive tool for evaluation of tumoral response to ICI. high-risk progressing neuroblastomas using RNA-sequencing
Primarily used in analysis of immunologic transcriptomic data led to the development of the immune-predictive score.56
patterns, multiple high-throughput genomic tests have been This utilizes 15 pairwise comparisons between immune
developed to predict sensitivity or resistance to checkpoint checkpoint expression profiles (including immunoinhibitory
inhibition. Among these signatures is the assessment of and immunostimulatory genes) and accurately predicts
expression patterns relating to IFN-γ and responsive genes. neuroblastoma response with an AUC of 0.9. Because of
possible correlations between immune regression in neuro-
For instance, in the POPLAR trial, pretreatment effector T-cell
blastoma and melanoma, immune-predictive score was
transcription patterns were analyzed in tumor samples of
validated among 10 independent cohorts of patients with
patients with progressive NSCLC receiving atezolizumab or
melanoma who were treated with ICI (AUC, 0.83); it also
docetaxel.51 These patterns incorporated expression levels of
correlated with PFS and OS. Similarly, transcriptomic patterns
CD8A, GZMA, GZMB, IFN-γ, EOMES, CXC motif chemokine
of tumoral immune escape have been used to predict patient
9 (CXCL9), CXCL10, and TBX21. OS was significantly im-
response to checkpoint inhibition. The tumor immune dys-
proved in patients receiving atezolizumab with high levels of
function and exclusion score uses Cox proportional hazards
this IFN-γ signature (HR, 0.43). Interestingly, PD-1/PD-L1
to compare gene expression signatures in tumor or immu-
pathway components were also assessed (including B7.1
nomodulatory cells with TIL levels.57 In cohorts with mela-
and PD-L2) and correlated with improved OS (HR, 0.46,
noma or NSCLC, this algorithm was more predictive of ICI
0.43, 0.45, and 0.39, respectively). In addition, subsequent
response than PD-L1 expression, IFN-γ signature, or TMB. It
data analyzing 18 genes (also largely IFN-γ–responsive tar-
also correlated strongly with PFS and OS among patients with
gets) among nine malignancies demonstrated a moderate
melanoma. Furthermore, the immunophenoscore has been
association with patient response to pembrolizumab.52 Im-
validated in two independent cohorts of patients with mel-
portantly, this “T-cell inflamed” GEP was prospectively vali-
anoma as being markedly superior (AUC, 1.0) in predicting
dated within the KEYNOTE-012 trial among patients with
response to checkpoint inhibition compared with TMB, cy-
HNSCC and was comparable to PD-L1 IHC predictive utility
tolytic score, or expression of CTLA-4, PD-L1, or PD-1.58 The
(area under the curve [AUC], 0.75 vs. 0.65).53 Furthermore,
score was derived using machine learning and incorporated
a smaller IFN-γ messenger RNA transcription pattern (in- metagene analysis unique to tumoral immune infiltrates with
corporating CD274, LAG3, CXCL9, and IFN-γ) was recently tumor genetic heterogeneity and clonality.
validated in a nonrandomized phase Ib/II clinical trial among
patients with NSCLC or urothelial cancer.54 Treatment with Multiplex immunohistochemistry and immunofluorescence
durvalumab among patients with elevated signature levels A unique approach to pathologic analysis of tumoral het-
was associated with higher overall response and longer erogeneity and treatment response is mIHC/IF. Multiple
median PFS and OS, also independent of PD-L1 IHC. In analytes within histologic preparations can be visualized
a retrospective collection of ICI-treated patients with mela- simultaneously with this technique and provide quantitative
noma, even single-gene (PD-L1) messenger RNA expression data about cellular spatial relationships. Earlier referenced
was associated with response.55 studies used this approach to determine the composition
and location of TILs among cohorts with melanoma dem-
An important concept within GEPs is that the predictive utility onstrating ICI response.47 Subsequent research has con-
of such algorithms may be dependent on individual therapy firmed the spatial density of PD-1 and PD-L1 within tumor
plans. For instance, recent studies evaluating the efficacy of parenchyma as a predictive biomarker. For instance, within
antihuman glucocorticoid-induced tumor necrosis factor re- a small discovery cohort of patients with metastatic mela-
ceptor antibodies among advanced solid malignancies failed noma, PD-1/PD-L1 proximity or coexpression of the major
to demonstrate predictability of IFN-γ GEPs to treatment.107 histocompatibility complex-II (HLA-DR) and indoleamine
This suggests that IFN-γ signaling and transcriptomic patterns 2,3-dioxygenase 1 was highly correlative with anti–PD-1
may correlate only with response to therapy of directly related response.59 Validation within a larger cohort demonstrated
targets (i.e., downstream PD-1/L1 inhibition). Unrelated im- that increases in these signatures were predictive of im-
mune pathways involving targets such as tumor necrosis provement in PFS (HR, 0.36; p = .0004) and OS (HR, 0.39;
factor-α or matrix metalloproteinase-9 may instead require p = .0011). Similar studies assessing PD-1 and PD-L1
separate and individualized gene expression assays. marker proximity in Merkel cell carcinoma reported that
One way to improve the accuracy of immune biomarkers, and they correlate positively with pembrolizumab response.60
to potentially make them applicable to more than one type of Another recent study used quantitative IF to assay TIL
therapy, is to develop comprehensive multifactor prediction activation and proliferation among patients with NSCLC.61
tools. One method is to evaluate the transcriptome profiles Increased coexpression of CD3, Ki-67, and granzyme B

among cytotoxic T cells was prognostic of improved survival elevation of peripheral CD27+CD45RO CD8+ T cells was
in ICI-naive patients. Interestingly, a “dormant” multiplex IF highly sensitive and specific for CR to tisagenlecleucel.68
signature demonstrating high CD3, but low granzyme B and Similarly, a subpopulation of CAR T-cell product (CD27+PD-
Ki-67, was significantly associated with durable clinical 1 CD8+) was highly predictive of treatment response (AUC,
benefit (86% rate), improved PFS, and improved OS fol- 0.92). Also, in a small study evaluating anti-CD19 CAR T-cell
lowing checkpoint inhibition. therapy among patients with B-cell leukemia or lymphoma,
These and other data clearly demonstrate the advantages of elevated pretreatment levels of interleukin (IL)-12 and den-
a multiplexed system over more traditional single-analyte dritic cell lysosome-associated membrane glycoprotein in
IHC/IF. As mentioned previously, systematic review and peripheral blood served as good predictors of positive re-
meta-analysis showed a high correlation between mIHC/IF sponse. Additionally, TNA-related apoptosis-inducing ligand
and patient response to anti–PD-1/L1 therapy (AUC, 0.79), and Fas ligand directly correlated with patient survival,
while outperforming biomarkers lsuch as PD-L1, TMB, and whereas IL-6, IL-8, PD-L1, PD-L2, and NAP3 were inversely
GEP (AUC, 0.65, 0.69, and 0.65, respectively).16 related.110 Another valuable metric for CAR T-cell therapy is
TMB and neoantigen formation. For instance, mutational
CHIMERIC ANTIGEN RECEPTOR T-CELL AND T-CELL load in related adoptive cell therapies holds promising pre-
ENGAGER BIOMARKERS dictive utility. In an early clinical phase trial among patients
A subset of adoptive cell therapy, CAR T-cell regimens with advanced and refractory melanoma, higher levels of
function by collecting autologous or allogeneic peripheral somatic mutations correlated with improved response, PFS,
T cells and selectively expressing synthetic receptors and OS to TIL transfer and IL-2.111 And several studies across
against tumoral antigens. Patients subsequently receive various cancer subtypes demonstrate improved efficacy of
infusions of these modified lymphocytes, which directly adoptive cell therapy when mutation-specific T-cell pop-
target cancerous cells and, ideally, maintain an active CAR ulations are expanded.112 Though these data do not directly
T-cell population for sustained surveillance. A major advent utilize therapy with CARs, clear practical extensions exist. A
in the treatment of relapsed and refractory hematologic broader mutational landscape and more diverse neoantigens
malignancies was the FDA approval of the anti-CD19 CAR increase the repertoire of putative CAR targets. This improves
T-cell products tisagenlecleucel and axicabtagene cil- the likelihood of developing truly tumor-specific CAR antigen
oleucel for the treatment of pediatric/adolescent and young combinations and overcoming a limitation behind the ma-
adult B-cell precursor ALL and adult large B-cell lymphoma, jority of current regimens: host tissue target expression and
respectively.65,108 The indications for tisagenlecleucel were unwanted toxicity.
quickly expanded to include cohorts with adult large B-cell Therapeutic targeting of effector T cells and innate immune
lymphoma.109 Multiple preclinical models and active clinical cells to tumor antigens can also be enhanced with the use of
trials are evaluating the efficacy of alternative synthetic bispecific monoclonal antibodies. Modified proteins with
receptors in diverse malignancies, including chronic lym- distinct antigen-binding fragments or fusion products be-
phocytic leukemia, multiple myeloma, mesothelioma, NSCLC, tween single-chain variable fragments serve to bridge im-
breast cancer, and glioblastoma, among others. mune cell receptors and cytotoxic activity with malignant
Although not as extensive or validated as biomarkers in cells.113 Despite rapid growth in the discovery and devel-
checkpoint inhibition, several quantitative patient measure- opment of these immunomodulatory formats, only one is
ments can predict response in CAR T-cell therapy. A major globally approved as a cancer therapeutic.114 Initial devel-
determinant of treatment success appears to be expansion opment of the trispecific antibody catumaxomab allowed for
and persistence of CAR T-cell populations following infusion. bridging among carcinoma-upregulated epithelial cell ad-
For example, among a small cohort of patients with advanced hesion molecules, CD3 receptors on T cells, and Fc-γ re-
B-cell ALL, anti-CD19 CAR T-cell therapy was associated with ceptors on accessory immune cells. This was approved in
an impressive CR (86%, negative minimal residual disease) by 2009 by the European Medicines Agency for the man-
day 28 postinfusion. However, loss of circulating CAR T cells agement of malignant ascites, although it has since been
by host CD8+ activity against the transgene product promoted removed from the market because of manufacturer in-
relapse among a subset of these responders.66 Correspond- solvency.115 Subsequently, the bispecific T-cell engager
ingly, in the ZUMA-1 trial, axicabtagene ciloleucel expansion antibody blinatumomab, targeting CD3 and CD19, was
was significantly associated with improved objective response approved by the FDA following the BLAST trial for use in
(peak factor 4.0); persistent serum levels were also demon- relapsed/refractory B-cell precursor ALL.116
strated in three patients with sustained CR at 24 months.65 Post hoc analysis of catumaxomab phase II/III testing
Immunophenotypes also serve as biomarkers for CAR T-cell demonstrated that increased pretherapy levels of peripheral
efficacy. For example, among high-risk and heavily treated blood relative lymphocytes (. 13%) improved mean OS
patients with chronic lymphocytic leukemia, pretherapy flow among patients treated with intraperitoneal catumaxomab

McKean et al
(131 days; p = .0072).69 Tregs are another biomarker that lower baseline levels of IL-6, the risk of ipilimumab toxicity
has been shown to correlate with bispecific-antibody re- was increased (odds ratio, 2.84; p = .007).73 Similarly,
sponse. Among a cohort of patients with B-cell precursor decreased pretreatment levels of circulating IL-6, IL-8,
ALL receiving blinatumomab, Treg levels in the peripheral soluble IL-2 receptor (sCD25), and IL-17 among cohorts
blood were increased among nonresponders (10.25%) with locally advanced or metastatic melanoma are associ-
compared with responders (4.82%).67 ated with ipilimumab-induced colitis.74,75 Interestingly, it
may be the net increase in these proinflammatory cytokines
BIOMARKERS AND ADVERSE EVENTS
from ICI therapy that determines immune toxicity. In two
The advent of modern cancer immunotherapeutics has been studies of melanoma cohorts treated with nivolumab, on-
accompanied by challenging side effect profiles requiring therapy increases in IL-676 or in soluble cluster of differ-
prompt and unique management. The clinical and financial entiation 163 (sCD163) and CXCL577 were predictive of
burden from these toxicities is significant. In ipilimumab psoriasiform dermatitis or diverse irAEs, respectively.
monotherapy alone, irAEs are seen in more than 70% of
Furthermore, the presence of autoantibodies may predict
patients.117,118 Combination checkpoint inhibition increases
development of endocrine-specific toxicity following check-
the incidence and onset of these irAEs; pooled analysis of
point inhibition. In a multivariate analysis of patients with
ipilimumab and nivolumab treatment among cohorts with
advanced solid tumors treated with nivolumab, pretreatment
melanoma suggests that nearly all patients (94%) experience
elevation of serum antithyroglobulin antibodies was signifi-
some degree of toxicity, with up to 55% of them being high
cantly associated with subsequent autoimmune thyroid
grade (Common Terminology Criteria for Adverse Events
dysfunction (odds ratio, 26.5; 95% CI, 8.18–85.8).78 Among
grades 3-4).119 Among patients with B-cell precursor ALL or
a separate cohort diagnosed with a variety of solid tumors,
diffuse large B-cell lymphoma treated with CAR T-cell therapy,
the presence of one or more diabetes autoantibodies (against
the development of CRS may increase hospitalization costs by
glutamic acid decarboxylase 65, islet antigen 2, insulin, islet
more than $50,000.120 Unfortunately, our understanding of
cells, or zinc transporter 8) prior to ICI therapy hastened the
biomarkers that are predictive for immunotherapy toxicity is
development of clinical diabetes.79
underdeveloped and warrants further investigation.121
PITFALLS OF CURRENT BIOMARKER USE
Cytokine Release Syndrome and Immune Effector
Cell–Associated Neurotoxicity Syndrome Biomarkers Although there is clear prognostic and predictive benefit
behind biomarkers of immunotherapy response and tox-
Predictive algorithms for the development of CAR T-cell
icity, significant limitations prohibit more widespread benefit
toxicity have been proposed in several studies.122 Among
among patients (Fig. 1). Dramatic variation within individual
cohorts of pediatric and adult patients with B-cell precursor
tumor parenchyma, chronologic change, microenviron-
ALL, forward-selected logistic regression or decision tree
ment, and sample preparation are each a potential source
models using combinations of cytokines accurately predicted
behind failure of treatment selection and response.
the development of severe (grades 4-5) CRS. 70 When
combining IFN-γ, IL-13, and macrophage inflammatory Intratumoral Heterogeneity
protein 1α in pediatric patients, sensitivity and specificity
Depending on the degree of diversity, subclonal populations
reached 100% and 96%, respectively. In a phase I/II clinical
within a tumor may not be adequately sampled with biopsy
trial of adult patients with advanced non-Hodgkin lymphoma,
alone (Fig. 1A). As such, preliminary analysis and biomarker
chronic lymphocytic leukemia, or B-cell precursor ALL,
testing from limited tissue may not be representative of
preinfusion increases in proteins associated with capillary
actual response to immunotherapy. Recent data published
leak (angiopoietin ratios and von Willebrand factor) correlated
on patients with NSCLC or melanoma suggest that subclonal
with increased severity of subsequent CRS.71 In this same
neoantigen heterogeneity is enriched in patients with poor
trial, classification-tree modeling demonstrated that elevated
response to ICI.123 This neoantigen diversity is more rep-
serum monocyte chemoattractant protein-1 levels ( 1343.5
resentative of patient outcomes than total neoantigen
pg/mL) and development of a fever 38.9°C or greater within
burden alone, and it can be combined with TMB to better
36 hours of CAR T-cell infusion were highly sensitive (100%)
stratify predictive data of median OS.
and specific (95%) for subsequent CRS (grade 4-5). Com-
bining elevated IL-6 concentrations ( 16 pg/mL) with this Mutational variety within a tumor may also limit the predictive
algorithm was also predictive of severe (grade 4-5) immune yield of certain biomarkers (Fig. 1D). For instance, although
effector cell–associated neurotoxicity syndrome.72 TMB shows broad correlation with patient survivorship and
therapy response in ICI, certain tumors with low TMB also
Immune-Related Adverse Event Biomarkers show increased responsiveness to checkpoint inhibition. This
Cytokines appear to be important markers of irAE risk as may be due to alternative mutations such as indels (seen at
well. For instance, among patients with melanoma and high frequency in renal cell carcinoma).15 Indeed, positive

FIGURE 1. Sources of Biomarker

Error
(A) Intratumoral (and intraper-
sonal) cellular heterogeneity re-
mains a significant limitation in
biomarker validity. Further dy-
namic alterations in clonal com-
position under the pressure of
time (1) and therapy (2) prohibit
pretreatment biomarker accu-
racy. (B) Patient host immunity
and surrounding tumor micro-
environment remain highly in-
dividualized and responsive to
progressive cytokine (1) and/or
treatment (2) exposures. These
multiple variables abrogate the
accuracy of single biomarker
tests. (C) HLA allelic polymorphism
and TCR selection generate sig-
nificant variety among patient
antigen processing and pre-
sentation. (D) Post-translational
protein modifications or novel
mutations such as indels and
copy number alterations gen-
erate neoantigens that may re-
main undetected by traditional
biomarkers.
Abbreviations: TMB, tumor mu-
tation burden; TAM, tumor-
associated macrophage; TIL,
tumor-infiltrating lymphocyte;
Treg, regulatory T cell; MDSC, myeloid-derived suppressor cell; O-GlcNAc, O-linked β-N-acetylglucosamine; TCR, T-cell receptor; MHC,
major histocompatibility complex.
Created with Biorender.
correlations with indel burden in cohorts with melanoma are predictors of patient response to immunotherapy (Fig. 1B).
associated with improved response to ICI therapy. Somatic For example, in several studies incorporating multiple
copy number alterations are another mutation type not cancer types, PD-L1 expression on tumor-infiltrating im-
currently assessed by TMB assays that have been positively mune cells (macrophages, dendritic cells, or T lympho-
associated with immune infiltration and survival advantage cytes) was correlative with ICI patient response, whereas
among cohorts with melanoma.36 Also, there is evidence that PD-L1 tumoral expression alone was not.25,48,49,126 Addi-
some neoantigens are generated by post-translational tionally, there is a growing body of evidence that tumor-
modifications, and these would not be detected by any associated macrophages may abrogate anti–PD-1 response
nucleic acid assay.124,125 in patient cohorts with advanced melanoma.12,127,128 Al-
ternatively, another unique cell population, myeloid-derived
Tumor Microenvironment suppressor cells, is inversely correlated with anti–CTLA-4
The complex interplay between tumor cells and their mi- response in patients with melanoma.129-132 Similarly, failure
croenvironment plays a significant role in patient response of adoptive T-cell therapy in solid tumors can be associated
to immunomodulatory agents. The proliferation or retardation directly with the proportion of myeloid-derived suppressor
of malignant tissue relies on multiple surrounding host cells; natural killer targeting of these cells improves results in
factors such as cytotoxicity of infiltrating lymphocytes, im- neuroblastoma xenograft models.133 These and other find-
munosuppression from tumor-associated macrophages, ings make it increasingly clear that nontumoral cells within
and paracrine signaling from supportive fibroblasts. As the microenvironment can affect biomarker utility and may
such, biomarkers of tumor cells alone may be insufficient serve as an underutilized predictive resource themselves.

McKean et al
Interpatient Variation and Host Immunity chemotherapy and immune therapy. With the generation of
Similar to intratumoral subclonal diversity, significant variation resistant subclones, the predictive power of static biomarkers
exists within histologic cancer types among separate patients. becomes more limited. For example, in patients with advanced
For instance, among patients with metastatic melanoma and melanoma, pretreatment TMB is positively associated with
naive to checkpoint inhibition, PD-L1 was homogenously anti–PD-1 survivorship only within therapy-naive cohorts.12
positive (defined as . 1% tumor expression) in 26% of the However, quantitative change in TMB during anti–PD-1
cohort and negative in 22% of subjects.134 Importantly, 52% of therapy strongly correlates with response and OS, even
patients showed intrapersonal PD-L1 heterogeneity among within patients previously treated with ICI. Similarly, on-therapy
primary melanoma sites, locoregional disease, and distant changes in T-cell diversity correlate with anti–PD-1 treatment
metastases, highlighting yet another limitation of current testing. outcomes. Among independent cohorts of patients with
melanoma naive to checkpoint inhibition, a dynamic loss in
Another level of this interpatient heterogeneity is the striking pretherapy TCR variation (and subsequent oligoclonal ex-
complexity within host antigen processing and presentation pansion) correlates positively with nivolumab response.12,142
(Fig. 1C). Significant polymorphism among HLA alleles—as However, this relationship is reversed among patients with
well as the extensive process behind cognate T-cell receptor progressive melanoma on anti–CTLA-4 treatment. Here,
(TCR) expression, recombination, and selection—generates nivolumab response correlates with increasing richness of the
remarkable individuality between patient immune systems. complementarity-determining region 3 on TCRs.12 Such
This variability, in turn, can influence patient outcomes to dramatic chronologic shifts among tumor and immune cells
checkpoint inhibition. For example, recent analysis of multiple require more frequent biomarker surveillance. Clearly, bi-
cancers among two cohorts demonstrated that maximal HLA opsies cannot be limited to single or pretreatment sampling.
loci heterozygosity improved OS after ICI therapy.135 Similarly,
loss of HLA-I heterozygosity correlated with reduced survival Sample Collection and Evaluation
after ICI therapy (HR, 1.60; p = .05) and was most pro- A major restriction in current biomarker use is the significant
nounced among patients with low TMB (HR, 3.68; p = .0006). variability in preanalytical processing and subsequent clinical
Within this same study, distinct supertype alleles had dis- interpretation. Depending on the laboratory test used, several
parate associations with patient outcomes after ICI. HLA-B44 independent factors are prone to error.143 For example,
expression was seen in patients with extended survival (HR, 0. background signal and morphologic clarity of IHC/IF can be
61; p = .01), whereas the presence of HLA-B62 had the influenced by sample fixation time, quality of embedding, or
opposite correlation (HR, 2.29; p = .0007). Extensive work has time from extraction to preservation. Similarly, the quality of
demonstrated that aberrant tumoral regulation or mutation of biomarkers in peripheral blood can be altered by cryopres-
HLA-I genes contributes to immune evasion and poorer ervation temperature, repeated freeze-thaw cycles, antico-
clinical response.136,137 By extension, pretherapy abrogation agulant type, and processing delay. Although not always
of major histocompatibility complex class I components is controllable, the ramification of such variables in immuno-
predictive of ipilimumab resistance, whereas response to therapy is clear. In PD-L1 IHC alone, numerous diagnostic
nivolumab may require major histocompatibility complex assays are available but are not always interchangeable; thus,
class II expression and IFN-γ activation.138 they can yield disparate results, even among case-matched
In addition, systemic TCR sequence diversity may be related samples.144 Furthermore, analytical interpretation of pro-
to ICI response. For instance, in two separate studies eval- cessed samples can show significant variation. Although
uating small cohorts of patients with metastatic pancreatic good reproducibility is observed among tumor cells, pa-
adenocarcinoma139 or urothelial carcinoma,140 the presence thologists have demonstrated poor concordance in PD-L1
of pretherapy peripheral T-cell clonality predicted poorer OS scoring of immune cells in NSCLC samples.145 Tighter control
on ipilimumab and poorer PFS and OS on atezolizumab. The of preanalytical processing and greater concordance among
relationship between TIL TCR variability and ICI response is data reporting are needed for further biomarker development
less clear; some studies suggest pretherapy clonality as and validation in clinical trials.146
a marker for anti–PD-1 response,37,47 whereas another study IDEAL AND FUTURISTIC BIOMARKER USE
correlates post-therapy clones with positive NSCLC outcomes
on nivolumab.141 The variance in these findings highlights the Biomarkers will be the future foundation of treating all
complexity of interactions between host immunity and the cancers with immunotherapy. Given the complexity of the
tumoral milieu. Such complexity significantly limits the power immune system and its regulation, it is unlikely that any single
and interpretation of canonical biomarkers. component biomarker will be predictive enough for all pa-
tients or all types of immunotherapies. Therefore, ideal bio-
Chronologic Change markers will need to be multifaceted and comprehensive
Dynamic change within tumoral subpopulations can oc- and be able to guide clinicians about whether the patient
cur over time, especially under the selective pressure of will respond to immunotherapy, as well as which specific

immunotherapeutic agent(s) (e.g., checkpoint inhibitors, OPTIMAL CLINICAL TRIAL DESIGN FOR BIOMARKER UTILITY
immunostimulatory therapies, cellular therapies) are needed Prospective data are required for proper biomarker devel-
(Table 2). If no such biomarker is available, early on- opment. Therefore, all future clinical trials must be designed
treatment biomarkers that confirm that a patient is (or is to incorporate biomarkers to some degree. At a minimum,
not) responding to immunotherapy will be needed. This type pretreatment and on-treatment information (e.g., tissue or
of response detection will allow physicians to identify patients blood samples, raw data from images, or clinical de-
who are not benefiting from a specific immunotherapy early mographics) must be collected and stored for potential
enough in their course so that treatment can be switched/ exploratory biomarker analyses. Biopsy and blood collection
escalated prior to complications that are associated with upon progression should also be considered and can be
tumor progression. Ideally, such biomarkers would be non- critical to elucidate resistance mechanisms and guide the
invasive; therefore, the development of noninvasive assess- next line of therapy. This is especially important for early-
ments of changes in the tumor microenvironment will be phase trials of novel immunotherapies, because the bio-
needed.147 Examples of these include PET imaging of TILs or marker(s) for response and/or resistance will likely be
immune checkpoints, as well as systemic cytokine changes. therapy specific. Being able to identify potential biomarkers
TABLE 2. Design and Advantages of Ideal Biomarkers in Immunotherapy

Noninvasive Better sample accessibility using peripheral blood or imaging allows for closer monitoring of therapy
response or the development of treatment resistance
Time Sensitive Reliable and accessible preliminary testing, combined with regular and consistent on-therapy
sampling, could provide actionable results of chronologic tumoral changes
Multifactorial and Because of the complexity of interactions among tumor parenchyma, microenvironment, and host
Comprehensive immune response, it is unlikely that single analytes or tumor-specific values alone will be sufficient for
predictive capacity in immunotherapy
Therapy Specific Within precision medicine, comprehensive biomarkers could predict ideal initial immunotherapy class
and subtype (i.e., anti–PD-1/L1 vs. anti–CTLA-4) and direct subsequent changes should resistance
develop
Resistance and Toxicity Adaptive clinical trials and individualized patient therapy will require predictors of adverse events and
treatment refractoriness to appropriately prioritize therapeutic options

McKean et al
for novel therapies early in development will allow the ex- improve the proportion of patients who are likely to respond
pedited validation and clinical use of these biomarkers in to therapy, there is a significant need for biomarkers with
late-phase trials. Although understanding who will respond stronger predictive abilities. Given the complexity of the
to a specific treatment is of high interest, equal interest and immune system, single-marker biomarkers will likely be in-
effort should be placed into evaluating biomarkers of sufficient to adequately predict who will and will not respond
nonresponse or resistance. Indeed, understanding these to cancer immunotherapy. One potential way to mitigate this
negative metrics may identify the best therapies to use in is to develop biomarkers that allow for the early assessment of
specific patient populations. Once these biomarkers of immune activation within the tumor microenvironment (e.g.,
response and resistance are further developed, adaptive noninvasive assessment of TILs, alternative checkpoints)
clinical trials will be needed. Here, on-treatment biomarkers rather than pretreatment predictive biomarkers. These types
of early response or resistance can identify patients who do of early response assessment biomarkers would allow pa-
not benefit from a particular therapy but instead require tients to initiate single-agent treatments with low side effect
treatment changes or dose escalation. Another large hurdle profiles and subsequently identify patients who need esca-
in immunotherapy development is the heterogeneity of lations/changes in therapy prior to clinical progression and
resistance mechanisms between patients. Many trials have deterioration. Given the complexity of host immunity, another
negative results, not because the agents are not active, but
potential mechanism for developing immune biomarkers is to
because the signal is diluted. Using biomarkers to separate
use multimarker assessments. However, given the multitude
patients into different groups of similar resistance mecha-
of novel immunotherapies currently under investigation (e.g.,
nisms can be a solution to focus drug-development efforts.
targeting alternative checkpoints, the adenosine pathway,
CONCLUSIONS immunostimulatory molecules, cytokines), it is likely that no
Cancer immunotherapy, primarily ICI, can provide durable, single biomarker, or even biomarker signatures, will be
long-lasting responses for some patients with cancer; predictive for each type of cancer immunotherapy. Therefore,
however, these therapies are currently effective in only comprehensive immune profiling of individual tumors will
a small minority. Although currently available biomarkers likely be needed to develop predictive biomarkers that can
(primarily PD-L1 and IFN-γ gene signature panels) can accurately direct the selection of patient immunotherapy.
AFFILIATIONS CORRESPONDING AUTHOR

1
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT Siwen Hu-Lieskovan, MD, PhD, Huntsman Cancer Institute, University of
2
HonorHealth Research Institute, Scottsdale, AZ Utah, 2000 Circle of Hope Drive, HCI-RS-2703, Salt Lake City, UT
3
Department of Pathology, Yale University School of Medicine, New 84112; email: siwen.hu-lieskovan@hci.utah.edu.
Haven, CT
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
AND DATA AVAILABILITY STATEMENT
Disclosures provided by the authors and data availability statement (if
applicable) are available with this article at DOI https://doi.org/10.1200/
EDBK_280571.
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Biomarkers in Precision Cancer Immunotherapy

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Biomarkers in Precision Cancer Immunotherapy

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DEVELOPMENTAL THERAPEUTICS—IMMUNOTHERAPY

Biomarkers in Precision Cancer Immunotherapy:

INTRODUCTION Adoptive cell therapies have also shown sustained

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downregulate priming and activity of cytotoxic responses.

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TABLE 1. Characteristics and Clinical Correlates of Biomarkers in Cancer Immunotherapy

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TABLE 1. Characteristics and Clinical Correlates of Biomarkers in Cancer Immunotherapy (Continued)

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Gene Expression Proﬁles among immune-activating and immune-inhibitory states. For

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FIGURE 1. Sources of Biomarker

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TABLE 2. Design and Advantages of Ideal Biomarkers in Immunotherapy

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AFFILIATIONS CORRESPONDING AUTHOR

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