CAP Laboratory Improvement Programs
The Cancer Immunotherapy Biomarker Testing Landscape
Eric E. Walk, MD; Sophia L. Yohe, MD; Amy Beckman, MD; Andrew Schade, MD, PhD; Mary M. Zutter, MD;
John Pfeifer, MD, PhD; Anna B. Berry, MD; on behalf of the College of American Pathologists Personalized Health Care Committee
 Context.—Cancer immunotherapy provides unprece-
dented rates of durable clinical benefit to late-stage cancer
patients across many tumor types, but there remains a
critical need for biomarkers to accurately predict clinical
response. Although some cancer immunotherapy tests are
associated with approved therapies and considered vali-
dated, other biomarkers are still emerging and at various
states of clinical and translational exploration.
Objective.—To provide pathologists with a current and
practical update on the evolving field of cancer immuno-
therapy testing. The scientific background, clinical data,
and testing methodology for the following cancer immu-
notherapy biomarkers are reviewed: programmed death
ligand-1 (PD-L1), mismatch repair, microsatellite instabil-
ity, tumor mutational burden, polymerase d and e
mutations, cancer neoantigens, tumor-infiltrating lympho-
cytes, transcriptional signatures of immune responsiveness,
cancer immunotherapy resistance biomarkers, and the
microbiome.
Data Sources.—Selected scientific publications and
C ancer immunotherapy has revolutionized the field of
oncology by delivering unprecedented levels of durable
survival benefit for cancer patients, including some patients
with previously incurable late-stage disease. It is now widely
accepted that the human immune system, when properly
activated and in the absence of negative regulatory
Accepted for publication August 23, 2019.
Published online November 12, 2019.
From the Department of Medical & Scientific Affairs, Roche Tissue
Diagnostics, Tucson, Arizona (Dr Walk); the Department of
Laboratory Medicine and Pathology, University of Minnesota
Medical School, Minneapolis (Drs Yohe and Beckman); Diagnostic
and Experimental Pathology, Eli Lilly and Company, Indianapolis,
Indiana (Dr Schade); the Department of Pathology, Microbiology,
and Immunology, Vanderbilt University School of Medicine, Nash-
ville, Tennessee (Dr Zutter); the Department of Molecular Pathology
and Genomics, Swedish Cancer Institute, Seattle, Washington (Dr
Berry); and the Department of Pathology, Washington University
School of Medicine, St Louis, Missouri (Dr Pfeifer).
Drs Pfeifer and Berry are co–senior authors.
Dr Walk is an employee at Roche Tissue Diagnostics and has stock
ownership. Dr Schade is an employee at Eli Lilly and Company. The
other authors have no relevant financial interest in the products or
companies described in this article.
All authors are past or current members of the College of American
Pathologists Personalized Health Care Committee.
Corresponding author: Eric E. Walk, MD, Department of Medical
& Scientific Affairs, Roche Tissue Diagnostics, 1910 E Innovation
Park Dr, Tucson, AZ 85718 (email: eric.walk@roche.com).
706 Arch Pathol Lab Med—Vol 144, June 2020
clinical trial data representing the current field of cancer
immunotherapy.
Conclusions.—The cancer immunotherapy field, includ-
ing the use of biomarker testing to predict patient
response, is still in evolution. PD-L1, mismatch repair,
and microsatellite instability testing are helping to guide
the use of US Food and Drug Administration–approved
therapies, but there remains a need for better predictors of
response and resistance. Several categories of tumor and
patient characteristics underlying immune responsiveness
are emerging and may represent the next generation of
cancer immunotherapy predictive biomarkers. Pathologists
have important roles and responsibilities as the field of
cancer immunotherapy continues to develop, including
leadership of translational studies, exploration of novel
biomarkers, and the accurate and timely implementation
of newly approved and validated companion diagnostics.
(Arch Pathol Lab Med. 2020;144:706–724; doi: 10.5858/
arpa.2018-0584-CP)
mechanisms, can efficiently eradicate even widespread
metastatic cancer.1,2 This realization has had a transforma-
tive impact on the fields of oncology, radiology, and cancer
drug development, as evidenced by new first-line treatment
options, new criteria for radiologic response,3 and dramatic
shifts in pharmaceutical pipeline strategies. The US Food
and Drug Administration (FDA) has approved multiple
cancer immunotherapies for a wide range of cancer
indications (Table 1),4 and more than 2000 cancer immu-
notherapy agents are currently in clinical development.5
Despite all the enthusiasm and positive clinical outcome
data, however, cancer immunotherapy currently benefits
only a small subset of cancer patients—around 20% on
average across the cancer indications assessed through
clinical trials.6 Because not all patients respond to cancer
immunotherapy, and some experience serious adverse
immune reactions,7 biomarkers predicting efficacy are
critically needed both for current clinical care and to enable
and drive further progress in this rapidly advancing field.
Anatomic and molecular pathologists have the opportu-
nity to be at the center of the development, validation, and
clinical implementation of patient selection biomarkers for
cancer immunotherapy. Predictive immunotherapy bio-
markers such as programmed death ligand-1 (PD-L1)
immunohistochemistry (IHC), mismatch repair (MMR)
IHC, and microsatellite instability (MSI) testing are already
established as routine in many pathology laboratories
around the world. In addition, emerging cancer immuno-
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
therapy biomarkers such as tumor-infiltrating lymphocyte
(TIL) assessment and multiplexed assessment of the tumor
microenvironment are dependent on in situ cellular and
histopathologic interpretation. Taken together, it is clear
that optimal progress in the field of cancer immunotherapy
would benefit from, and, one could argue, is dependent
upon, pathologist leadership and stewardship.
Clear guidelines for cancer immunotherapy testing are not
currently available, and the field is evolving rapidly in
response to new clinical and translational data. The intent of
this article is to briefly review the current landscape of
cancer immunotherapy biomarker testing as a resource for
individualized test selection in clinical pathology practice
and to provide an overview of this complex, ever-changing
field. Included are those cancer immunotherapy tests
currently accepted as standard of care for clinical testing,
including those assays with FDA approval as well as
emerging assays and technologies still undergoing investi-
gation and clinical validation. For each biomarker, the
clinical adoption status, scientific background, available
clinical data, and testing methodology will be reviewed.
This article is focused on providing a general awareness of
the current testing landscape. It is not meant to be a
complete review of the entire cancer immunotherapy field,
nor is it meant to serve as an official guideline for
immunotherapy testing or to provide clinical treatment
recommendations.
BACKGROUND AND HISTORY OF CANCER
IMMUNOTHERAPY
The immune system is divided into 2 distinct response
components, innate and adaptive. The innate immune
system responds rapidly but indiscriminately and includes
neutrophils and macrophages, whose receptors recognize
foreign antigens such as microbial products. In contrast, the
adaptive immune response is highly targeted and precise,
with a vast antigen response diversity achieved through the
rearrangement of B-lymphocyte immunoglobulin genes and
T-lymphocyte antigen receptor genes.8 Although slower to
develop, the incredible selectivity of the adaptive immune
system appears to underlie the clinical effectiveness of
cancer immunotherapy.
Although associations between infections and cancer
regression had been made as early as the 1700s, William
Coley, MD, is widely acknowledged as the ‘‘Father of
Immunotherapy’’ because of his systematic, lifelong study of
induced bacterial infections as therapy for sarcoma patients,
beginning in 1891.9 Although Coley firmly believed in the
tie between severe infections and cancer regressions, the
mechanisms underlying the responses he observed were not
elucidated until the mid to late 1950s with the realization
that the immune system could recognize and attack tumors
based on specific antigens.10,11
Our current mechanistic understanding of immune-medi-
ated cancer elimination is based on the widely accepted
process referred to as the cancer immunity cycle (Figure 1).12
This process, which is dependent on the adaptive immune
system, is initiated when necrotic and/or apoptotic tumor
cells release immunogenic neoantigen proteins into the
tumor microenvironment. These neoantigens are recognized
as foreign by the immune system, and are engulfed and
processed by dendritic cells, which migrate through lym-
phatics to lymph nodes to prime and activate tumor-specific
cytotoxic T-cell responses. Tumor-specific CD8þ cytotoxic T
Arch Pathol Lab Med—Vol 144, June 2020
cells then traffic back to the tumor to find and destroy their
cancer cell targets, triggering release of additional tumor
neoantigens and renewing the cancer immunity cycle.
Interruption of the cancer immunity cycle can clearly
occur and results in immune evasion, uncontrolled tumor
growth, and clinical progression. The success of cancer
immunotherapy to date has come from a careful elucidation
of this cycle, including the specific parameters and
mechanisms underlying immune responsiveness and resis-
tance, an approach that will continue to be necessary for
further advancement of the field. The first of the cancer
immune mechanisms to be understood and successfully
targeted were the immune regulatory checkpoints.
A complex system of regulatory checkpoints has evolved in
humans to ensure immune homeostasis and prevent
autoimmunity. Central to this system of regulation are
immune checkpoint proteins present on T lymphocytes and
antigen-presenting cells. More than a dozen immune
checkpoint proteins have been discovered, some functioning
to activate the immune system and others functioning to
down-regulate it.13 The pioneering work of Jim Allison, PhD
(MD Anderson Cancer Center, Houston, Texas), led to the
discovery that cytotoxic T-lymphocyte associated protein 4
(CTLA-4) acts as an inhibitory checkpoint molecule, func-
tioning as a brake on antitumor immune responses.14 Dr
Allison hypothesized that inhibiting CTLA-4 via antibody
blockade would abrogate suppression of immune priming
and enhance the immune response to cancer.14 Initial positive
results in animals led to subsequent clinical validation of
CTLA-4 inhibition in metastatic melanoma and to eventual
FDA approval of ipilimumab in 2011.15 Dr Allison and Tasuku
Honjo, MD, PhD (Kyoto University, Kyoto, Japan) were
jointly awarded the 2018 Nobel Prize in Physiology or
Medicine for their discovery of cancer therapy by inhibition of
negative immune regulation.16 In 2014, the FDA approved
nivolumab, which inhibits the programmed death receptor-1
(PD-1), a checkpoint molecule that directly regulates T-cell
effector activity in the tumor microenvironment. There are
currently several FDA-approved checkpoint inhibitor thera-
pies targeting CTLA-4 as well as PD-1 and its ligand, PD-L1
(Table 1),17,18 with numerous additional immune checkpoint
inhibitors in clinical development.5
PD-L1 (STATUS: FDA-APPROVED THERAPIES AND
ASSAYS)
PD-L1 Biology and Anti–PD-(L)1 Therapies
The PD-L1 ligand and its cognate receptor, PD-1, are
components of an immune inhibitory axis that, when
activated, negatively regulates T-cell signaling, effector
function, and killing capacity.13 Tumor cells can activate this
pathway through expression of PD-L1 on the cell surface via
2 distinct mechanisms, a constitutive mechanism triggered by
genomic alterations (see Table 2) and an inducible mecha-
nism termed adaptive immune resistance.13,19 In the latter case,
tumor surface expression of PD-L1 occurs in response to the
release of the cytokine interferon c (IFN-c) from T cells upon
tumor recognition. In this way, tumors avoid immune-
mediated destruction by activating a naturally occurring
immune regulatory pathway. Reactivation of T-cell prolifer-
ation and effector function through antibody blockade of PD-
1/PD-L1 has been conclusively shown to yield durable
clinical benefit to patients across multiple tumor types.20
Five anti–PD-(L)1 therapies (atezolizumab, avelumab,
durvalumab, nivolumab, and pembrolizumab) have been
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 707
 Table 1. FDA-Approved Cancer Immunotherapies
Drug Indication
Drug Target Indication Stage Line of Therapy Diagnostic
Checkpoint inhibitors
Atezolizumab in combination PD-L1 Nonsquamous NSCLC with no EGFR Metastatic First None
with bevacizumab, or ALK genomic tumor aberrations
paclitaxel, and carboplatin
Atezolizumab PD-L1 NSCLC Metastatic Second None
Urothelial carcinoma (aa) Locally advanced or metastatic First in cisplatin ineligible FDA-approved
urothelial carcinoma PD-L1 IHC
First in platinum (any) ineligible None
Second after any platinum None
Atezolizumab in combination PD-L1 Small cell lung cancer Extensive stage First None
with carboplatin and
etoposide

708 Arch Pathol Lab Med—Vol 144, June 2020

Atezolizumab in combination PD-L1 Triple-negative breast cancer (aa) Unresectable locally advanced or First FDA-approved
with paclitaxel protein-bound metastatic PD-L1 IHC
Avelumab PD-L1 Merkel cell carcinoma (aa) Metastatic First, any None
Urothelial carcinoma (aa) Locally advanced or metastatic Second
Durvalumab PD-L1 Urothelial carcinoma (aa) Locally advanced or metastatic Second None
NSCLC Unresectable stage III that has not progressed following concurrent
platinum-based chemotherapy and radiation therapy
Ipilimumab CTLA-4 Melanoma Metastatic First None
Cutaneous melanoma, completely Lymph node positive Adjuvant
resected
Ipilimumab þ nivolumab CTLA-4 Melanoma Unresectable or metastatic First None
þ PD-1 Colorectal cancer, dMMR or MSI Metastatic Second dMMR or MSI
high (aa) high
RCC, intermediate or poor risk, Advanced First None
previously untreated
Nivolumab PD-1 Classical Hodgkin lymphoma (aa) Any Second after autologous HSCT and None
brentuximab vedotin, fourth after 3
or more lines of systemic therapy
that includes autologous HSCT
Colorectal cancer, dMMR or MSI Metastatic Second dMMR or MSI
high (aa) high
Hepatocellular carcinoma (aa) Any Second, previously treated with None
sorafenib
Melanoma Unresectable or metastatic First
Melanoma, completely resected Involvement of lymph nodes or Adjuvant
metastatic
NSCLC Metastatic Second
RCC Advanced Second after prior antiangiogenic
therapy
SCCHN Recurrent or metastatic Second
Small cell lung cancer (aa) Metastatic Second
Urothelial carcinoma (aa) Locally advanced or metastatic Second

Cancer Immunotherapy Biomarker Testing Landscape—Walk et al

 Table 1. Continued
Drug Indication
Drug Target Indication Stage Line of Therapy Diagnostic
Pembrolizumab PD-1 Cervical cancer (aa) Recurrent or metastatic Second PD-L1 by FDA-
approved test
Classical Hodgkin lymphoma (aa) Refractory Fourth None
Esophageal squamous cell carcinoma Recurrent locally advanced or Second PD-L1 by FDA-
metastatic approved test
Gastric or GEJ adenocarcinoma (aa) Recurrent locally advanced or Third PD-L1 by FDA-
metastatic approved test
Hepatocellular carcinoma (aa) Previously treated with sorafenib None
HNSCC (aa) Metastatic Second None
Melanoma Unresectable or metastatic First, any

Arch Pathol Lab Med—Vol 144, June 2020

Melanoma Adjuvant treatment of patients with melanoma with involvement of lymph
node(s) following complete resection
Merkel cell carcinoma (aa) Recurrent locally advanced or First
metastatic
NSCLC with no EGFR or ALK Stage III ineligible for surgical First PD-L1 by FDA-
genomic tumor aberrations resection or definitive approved test
chemoradiation or metastatic
NSCLC Metastatic Second PD-L1 by FDA-
approved test
Primary mediastinal large B-cell Refractory Third None
lymphoma (aa)
Solid tumors, dMMR or MSI high (aa) Unresectable or metastatic Second dMMR or MSI
high
Urothelial carcinoma (aa) Locally advanced or metastatic First in cisplatin ineligible FDA-approved
PD-L1 IHC
First in platinum (any) ineligible None
Second None
Pembrolizumab in combination PD-1 RCC Advanced First None
with axitinib
Pembrolizumab in combination PD-1 Nonsquamous NSCLC with no EGFR Metastatic First None
with pemetrexed and or ALK genomic tumor aberrations
platinum chemotherapy
Pembrolizumab in combination PD-1 Squamous NSCLC Metastatic First None
with carboplatin and either
paclitaxel or paclitaxel
protein bound
Chimeric antigen receptor T-cell therapies
Axicabtagene ciloleucel CD19 Large B-cell lymphoma Relapsed/refractory Third None
Tisagenlecleucel CD19 Pediatric and young adult B-cell Relapsed/refractory Relapsed/refractory None
precursor ALL
Adult diffuse large-B cell lymphoma Relapsed/refractory Third
Abbreviations: aa, indication approved under FDA-accelerated approval (continued approval may be contingent upon verification and description of clinical benefit in confirmatory trials); ALK,
anaplastic lymphoma kinase; ALL, acute lymphoblastic leukemia; CTLA-4, cytotoxic T-lymphocyte associated protein 4; dMMR, mismatch repair deficient; EGFR, epidermal growth factor receptor;
FDA, US Food & Drug Administration; GEJ, gastroesophageal junction; HNSCC, squamous cell carcinoma of the head and neck; HSCT, hematopoietic stem cell transplantation; IHC,
immunohistochemistry; MSI, microsatellite instability; NSCLC, non–small cell lung carcinoma; PD-1, programmed death receptor-1; PD-L1, programmed death ligand-1; RCC, renal cell carcinoma;

Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 709

SCCHN, squamous cell carcinoma of the head and neck.
Figure 1. The cancer immunity cycle. Adapted from Chen DS, Mellman I.12 Oncology meets immunology: the cancer-immunity cycle. Immunity.
2013;39(1):1–10.

approved by one or more global regulatory agencies across
multiple cancer indications (Table 1).4 A recent systematic
review and meta-analysis by Khunger et al21 examined 41
PD-1/PD-L1 inhibitor clinical trials and found that PD-L1
IHC positivity in tumor cells and immune cells was
predictive of favorable response across all tumor types.
Generally, PD-L1 expression is associated with a greater
likelihood of benefit from anti–PD-(L)1 therapy, but, in
contrast to traditional companion diagnostic markers such
as HER2 or ALK, patients with PD-L1–negative tumors also
benefit from these therapies up to 20% of the time,
depending on the specific study, therapy, and indication.22–24
In response to these data, the FDA created a novel
diagnostic category termed complementary diagnostic, defined
(in a draft definition given at the 2016 American Society of
Clinical Oncology annual meeting) as ‘‘a test that aids in the
benefit-risk decision-making about the use of the thera-
peutic product, where the difference in benefit-risk is
clinically meaningful.’’25 In contrast, the FDA defines a
companion diagnostic as an ‘‘in vitro diagnostic device or an
imaging tool that provides information that is essential for
the safe and effective use of a corresponding therapeutic
product.’’26
PD-L1 Testing
Four separate PD-L1 assays27–30 have been developed and
approved in association with the clinical development of the
different FDA-approved anti–PD-(L)1 therapies and cancer
indications, leading to distinct therapy-indication-test com-
binations (Table 3). In addition, a fifth assay based on the

Table 2. Alternative Mechanisms of Programmed Death Ligand-1 (PD-L1) Expression

Mechanism
PTEN deletion, PI3K activation
ALK rearrangements
PD-L1 amplification/copy
number alteration
PD-L1 gene structural variations
disrupting the 3 0 UTR
Stabilization of PD-L1 mRNA
Abbreviations: ALK, anaplastic lymphoma kinase; mRNA, messenger RNA; UTR, untranslated region.
Findings Source, y
PTEN deletion and activation of the PI3K pathway increases PD-L1 Parsa et al,180 2007
expression in glioblastoma
ALK rearrangements have been shown to induce PD-L1 expression in Marzec et al,181 2008
anaplastic large cell lymphoma and non–small cell lung cancer Ota et al,182 2015
PD-L1 amplification/copy number alteration has been identified in multiple Roemer et al, 183 2016
tumor types, including 97% of classic Hodgkin lymphomas and more Chapuy et al,184 2016
than 50% of B-cell lymphomas
Goodman et al,185 2018
PD-L1 3 0 UTR may serve as a genetic marker for identifying cancers that Kataoka et al,186 2016
actively evade immune surveillance and may potentially respond to
checkpoint inhibition
Oncogenic RAS signaling can upregulate tumor cell PD-L1 expression Coelho et al,187 2017
through a mechanism involving increases in PD-L1 mRNA stability via
modulation of the AU-rich element-binding protein tristetraprolin (TTP)

710 Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
 Table 3. Approved Programmed Death Ligand-1 (PD-L1) Immunohistochemistry (IHC) Assays
EMA
Assay Name Indication Drug PD-L1 Scoring Measure Indication-Specific Cutoff FDA Classification Classification
PD-L1 IHC 22C3 NSCLC, stage III, first line Pembrolizumab TPS TPS  1% PMA: companion CE mark
PharmDx NSCLC, metastatic, second line TPS TPS  1%
Gastric or GEJ adenocarcinoma, CPS CPS  1

Arch Pathol Lab Med—Vol 144, June 2020

recurrent locally advanced or
metastatic
Cervical cancer, recurrent or CPS  1
metastatic
UC, locally advanced or CPS  10
metastatic
Esophageal squamous cell CPS  10
carcinoma, recurrent, locally
advanced or metastatic
PD-L1 IHC 28-8 Nonsquamous NSCLC Nivolumab % evaluable tumor cells 1%, 5%, 10% PMA: complementary CE mark
PharmDx Melanoma exhibiting partial or 1%
complete membrane
SCCHN 1%
staining at any intensity
UC 1%
Ventana PD-L1 (SP142) UC—first line, cisplatin ineligible Atezolizumab IC 5% IC PMA: companion CE mark
assay NSCLC TC, IC 50% TC or 10% IC PMA: complementary
TNBC Atezolizumab IC 1% IC PMA: companion
Ventana PD-L1 (SP263) UC Durvalumab % tumor cells with any 25% of tumor cells exhibit PMA: complementary CE mark
assay membrane staining membrane staining; or
above background, ICþ ICP . 1% and ICþ  25%; or
ICP ¼ 1% and ICþ ¼ 100%
NSCLC—first line Pembrolizumab TC 50% NA CE mark
NSCLC—second line 1%
NSCLC—second line Nivolumab 1%, 5%, 10%
Abbreviations: CPS, combined positive score, the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100; CE,
European conformity; EMA, European Medicines Agency; FDA, US Food & Drug Administration; GEJ, gastroesophageal junction; IC, the proportion of tumor area occupied by PD-L1 expressing tumor-
infiltrating immune cells of any intensity; ICþ, the percentage of tumor-associated immune cells with staining at any intensity above background; ICP, immune cells present, the percentage of tumor area
occupied by any tumor-associated immune cells; NA, Not applicable; NSCLC, non–small cell lung carcinoma; PMA, premarket approval; SCCHN, squamous cell carcinoma of the head and neck; TC,
percentage of tumor cells with PD-L1 membrane staining at any intensity above background staining as noted on the corresponding negative control; TNBC, triple-negative breast cancer; TPS, tumor
proportion score, the percentage of viable tumor cells showing partial or complete membrane staining at any intensity; UC, urothelial carcinoma.

Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 711

73-10 PD-L1 clone is currently in development in associ-
ation with the PD-L1 inhibitor avelumab. This is a rapidly
evolving field, however, and changes to the current PD-L1
testing landscape are expected with approvals of additional
immunotherapies, therapeutic indications, and assays.
Based on each combination of assay, therapy, and indica-
tion, the PD-L1 scoring measure and threshold for positivity
(ie, cutoff) can vary. For example, the companion diagnostic
assay based on the 22C3 clone for the pembrolizumab non–
small cell lung cancer (NSCLC) first-line indication uses a
tumor proportion score with a 50% cutoff, whereas the
companion diagnostic assay based on the SP142 clone for
atezolizumab in the triple-negative breast cancer indication
assesses tumor-infiltrating immune cells at a 1% cutoff
(Table 3). The most current version of the manufacturer’s
package insert and/or the interpretation guide for each assay
should be used for clinical testing and scoring. In addition to
the FDA–approved assays, nonapproved PD-L1 antibody
clones have been independently developed as laboratory-
developed tests, also known as laboratory-developed assays,
per College of American Pathologists and Clinical Labora-
tory Improvement Amendments of 1988 guidelines.31,32 A
2017 comprehensive review of PD-L1 laboratory-developed
assays demonstrated variable concordance between them,
concluding that standardization is needed before PD-L1
laboratory-developed assays can be recommended for
routine clinical use.33 Because FDA labeling of PD-1/PD-
L1 immunotherapies with an associated companion diag-
nostic requires tumors to be tested for PD-L1 as determined
by an FDA-approved test, use of PD-L1 laboratory-
developed tests in these indications would be considered
off-label use of the respective therapy.34,35
Based on the associated therapeutic efficacy and safety
data, the FDA has designated PD-L1 as a complementary
diagnostic for some specific PD-(L)1 inhibitor indication
intended uses and as a companion diagnostic for others
(Table 3). In addition, the FDA has demonstrated that it will
adapt drug and diagnostic labels based on emerging clinical
and safety data. For example, in June 2018, the FDA
restricted the use of pembrolizumab and atezolizumab in
patients with locally advanced or metastatic urothelial
cancer to those whose tumors express PD-L1 and who are
not eligible for cisplatin-containing therapy. This restriction
was based on the finding in 2 ongoing clinical trials that PD-
L1–low patients treated with anti–PD-(L)1 therapy had
decreased survival compared with patients receiving che-
motherapy.36 The labels of both drugs and the associated
diagnostic assays were revised accordingly, thereby con-
verting PD-L1 from an optional complementary diagnostic
to a mandatory companion diagnostic for this specific
clinical indication.
Several studies have assessed the analytic and technical
concordance between the on-market PD-L1 IHC assays.
The Blueprint Project was an industry-academic partnership
sponsored by the International Association for the Study of
Lung Cancer designed to provide information on the
analytic comparability of the various PD-L1 assays. Phase
1 of the project examined 39 NSCLC cases scored by 3
pathologists across 4 PD-L1 antibody clones (22c3, 28-8,
SP142, and SP263).37 The results demonstrated comparable
tumor cell staining among the 22c3, 28-8, and SP263 assays,
with the SP142 assay exhibiting fewer stained tumor cells
relative to the other 3 assays. Variability of immune cell
staining was greater than for tumor cell staining across all
assays, and in 37% of cases (14 of 38), a different PD-L1
712 Arch Pathol Lab Med—Vol 144, June 2020
classification would have been made depending on which
assay and cutoff combination was used. Extending the
comparison to 81 additional lung cancer cases, phase 2 of
the project validated the results of phase 1, with the 22C3,
28-8, and SP263 assays again demonstrating highly
comparable staining results and the SP-142 assay demon-
strating a lower proportion of tumor cell staining.38
Additionally, the 73-10 assay was found to have a higher
PD-L1 analytic sensitivity versus the other assays. Intra-
reader reproducibility was high for tumor cell PD-L1 scoring
(intraclass correlation coefficient, 0.86–0.93) but low (intra-
class correlation coefficient, 0.18–0.19) for immune cell
scoring. The study also found a high concordance between
PD-L1 scoring using glass slides versus digital images
(Pearson correlation .0.96) and good agreement in
assessing PD-L1 in cytology cell block material (intraclass
correlation coefficient, 0.78–0.85).
Rimm et al39 conducted a prospective, multi-institutional,
pathologist-based assessment of 28-8, 22c3, SP142, and
E1L3N PD-L1 IHC assays (the SP263 PD-L1 assay was not
included in this comparison). The study, which was
sponsored by the National Comprehensive Cancer Network
and funded by Bristol-Myers Squibb, found that the 22c3-
based assay showed a slightly but statistically significantly
lower staining level than the 28-8 and E1L3N assays, but the
authors ultimately concluded that the 28-8, 22c3, and
E1L3N assays are ‘‘essentially equivalent.’’ Consistent with
the Blueprint project findings, the SP142 assay was
associated with a significantly lower mean PD-L1 expres-
sion score in tumor cells. The study also found that
interreader reproducibility across assays was excellent for
tumor cell PD-L1 expression, but poor for immune cell
expression.
Recently, Munari et al40 conducted an interclone compar-
ison of the 22c3 and SP263 PD-L1 assays on 198 NSCLC
cases, concluding that the 2 assays are not interchangeable,
with statistically significant differences seen at both the 1%
and 50% cutoffs. At the 50% cutoff, approximately half of
the cases positive for SP263 would have been interpreted as
negative for 22c3. Although the authors were not able to
perform a direct correlation with response to anti–PD-(L)1
therapy, they concluded that these differences could lead to
‘‘possible underestimation of patients suitable for pembro-
lizumab therapy.’’
Although these analytic comparison studies are valuable,
no study to date has examined cross-assay concordance
against clinical outcome endpoint measures such as
response rate, progression-free survival, or overall survival
in a common PD-(L)1 therapeutic trial. Assessment and
validation of multiple PD-L1 assays and cutoffs across
multiple PD-L1 inhibitors would allow a determination of
true clinical sensitivity and specificity and therefore repre-
sents a priority in the field of cancer immunotherapy.
PD-L1 heterogeneity, both intratumoral41 and intertu-
moral,42,43 has been described and represents a challenge for
reliably assessing PD-L1 in the diagnostic setting. Ilie et al44
performed a comparative study of the PD-L1 status between
surgically resected specimens and matched biopsies of
NSCLC patients. They found a discordance rate of
approximately 50%, with the majority of the discrepancies
showing PD-L1–negative biopsies and PD-L1–positive
surgical resections from the same patient (75% of the
discordances were due to differences in immune cell
scoring). Therefore, PD-L1–negative tumors that respond
to immune checkpoint inhibitors may represent heteroge-
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
Figure 2. The frequency of microsatellite instability–high (MSI-H) status of 7919 tumor and matched normal pairs across 23 tumor types. Adapted
from Cortes-Ciriano I et al.56 Nat Commun. 2017;8:15180.
neous and/or temporal expression that was not detected, or
potentially not present, at the time of initial specimen
testing.45 Radiation therapy, chemotherapy, and small-
molecule kinase inhibitors have all been shown to induce
PD-L1 expression46–48 and therefore could represent bio-
logical causes of pretreatment versus posttreatment PD-L1
testing discrepancy.
MMR AND MSI (STATUS: FDA-APPROVED THERAPIES
REQUIRE TESTING)
MMR Pathobiology
The DNA MMR system recognizes and corrects insertion,
deletion, and base pair mismatches that occur during DNA
replication.49 Deficiencies in MMR are primarily caused by
inactivation of one or more of the 4 main proteins: MLH1,
MSH2, PMS2, and MSH6. Mismatch repair deficiency
(dMMR) can be inherited or acquired (sporadic) and leads
to accumulation of 2 main types of mutations in the DNA:
missense mutations throughout the genome and changes in
the length of microsatellite regions.50,51
Although inherited MMR defects can be caused by
inactivating mutations of any of the 4 MMR genes, MLH1
and MSH2 mutations account for approximately 90% of
cases, with mutations in PMS2 and MSH6 accounting for
most of the remaining cases.52,53 Sporadic MMR defects are
Arch Pathol Lab Med—Vol 144, June 2020
usually caused by epigenetic silencing of MLH1 via
promoter methylation and are frequently associated with
BRAF V600E mutations.54,55
Mismatch repair deficiency was first detected in colorectal
cancer (CRC) but can occur in many other tumor types56,57
(Figure 2), with a prevalence of 4% across all adult solid
malignancies.58 Tumors with a significant frequency of
dMMR include endometrial, gastric, small intestinal, colo-
rectal, cervical, prostate, bile duct, liver, and thyroid
carcinomas; neuroendocrine tumors; and uterine sarco-
mas.58 In one study, endometrial, gastric, and small
intestinal cancers were more likely to have MMR defects
than colon cancer.59
Inherited deficiencies of MMR are the cause of hereditary
nonpolyposis colon cancer, also known as Lynch syndrome,
a genetic syndrome with a predisposition to cancer,
especially CRC and endometrial carcinoma (52%–82% and
25%–60% lifetime risk, respectively). Approximately 8%–
16% of dMMR CRC cases are associated with Lynch
syndrome, with the remaining cases representing sporadic
CRC.60–63 Identifying cases of Lynch syndrome is critical
given the increased cancer risk not only for the patient (ie,
for other tumors), but also for the patient’s family members.
Therefore, it is recommended that all cases of CRC and
endometrial carcinoma undergo screening for MMR de-
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 713
fects.64–66 If dMMR is detected, further testing should be
performed to determine if the defect is inherited or sporadic
after the appropriate genetic counseling and consent is
obtained.
MSI Pathobiology
Microsatellites, also known as short tandem repeats or
simple sequence repeats,67,68 are short repetitive DNA
sequences that occur commonly throughout the genome,
primarily within noncoding intergenic regions and introns.
During DNA replication, transient dissociation and subse-
quent misaligned reassociation of the replicating DNA
strands can cause alterations in the length of these
microsatellites. These alterations are usually corrected by
the MMR system, but in the setting of dMMR can remain
uncorrected, resulting in MSI.69 Therefore, MSI is a genomic
consequence of dMMR, with the severity of instability
stratified by the number of affected microsatellite markers
into 3 groups: MSI high (MSI-H), MSI low, and microsat-
ellite stable (see MMR and MSI Testing Methods below).69
MMR, MSI, and Cancer Immunotherapy
Testing for dMMR and MSI has become a mandatory
component of identifying patients most likely to respond to
immunotherapy targeting PD-(L)1 in certain indications.
The first clinical observation suggesting that MMR status
predicted clinical response to anti–PD-1 blockade came
from a phase 2 study of the PD-1 checkpoint inhibitor
pembrolizumab.70 Le et al70 hypothesized that the efficacy
rate of PD-1 blockade in CRC was associated with MMR
status and initiated a phase 2 clinical trial to evaluate
pembrolizumab in patients whose tumors were dMMR or
MMR proficient (pMMR). The results of this study showed a
dramatic difference in the objective response rate (ORR) of
dMMR versus pMMR patients, with a 40% ORR in the
dMMR CRC patients versus 0% ORR in the pMMR CRC
patients. Additionally, dMMR non-CRC patients, including
those with ampullary, endometrial, small bowel, and gastric
cancers, also experienced a high rate of benefit with a 71%
ORR.
Currently, 2 cancer immunotherapy drugs are FDA
approved for dMMR tumors based on IHC or MSI
polymerase chain reaction (PCR) testing. In May 2017, the
FDA granted accelerated approval to pembrolizumab for
treatment of unresectable or metastatic dMMR or MSI-H
solid tumors that have progressed following prior treatment,
the first cancer site–agnostic approval granted from the
FDA.71 The data for pembrolizumab were based on the
results from 5 single-arm multicenter trials (KEYNOTE-016,
-164, -012, -028, and -158) of 149 total patients (90 CRC
patients, 59 with 14 other cancer types) showing an ORR of
40%, which was similar irrespective of tumor type.71 In these
studies, determination of MSI-H or dMMR status for the
majority of patients was prospectively performed via PCR
for MSI-H status or IHC for dMMR. The second FDA
approval related to MMR deficiency occurred when nivolu-
mab was approved in July 2017 for CRC that has progressed
following therapy, based on the CheckMate 142 study of 53
CRC patients showing an ORR of 32% in the overall
population.72,73
The association between dMMR response to cancer
immunotherapy appears to be driven by the probabilistic
creation of nonself, cancer-specific antigens termed neo-
antigens (see the Cancer Neoantigens section). These
neoantigens, in the presence of a cancer immunotherapy–
714 Arch Pathol Lab Med—Vol 144, June 2020
enabled activated T-cell immune response, are sufficient to
elicit antitumor immunity. Mismatch repair deficiency is
simply one mechanism that can create increased tumor
mutations, thereby increasing the probability of a neo-
antigen that can be processed and recognized by the
adaptive immune system. For example, dMMR tumors
harbor a 10- to 100-fold higher rate of mutations compared
with pMMR tumors.74,75 High tumor mutational burden
(TMB) (see the TMB section) can be created via other defects
in the DNA editing machinery, such as loss of polymerase
exonuclease function via POLE and POLD1 mutations (see
the POLE and POLD1 Mutations section).
MMR and MSI Testing Methods
There are 2 main methods of screening for MMR
functional defects: IHC for the 4 MMR proteins MLH1,
MSH2, PMS2, and MSH6,76,77 and molecular PCR testing to
detect MSI.78–80 Each method has unique limitations and
diagnostic pitfalls, with IHC being dependent on fixation
conditions and accurate interpretation and MSI requiring
normal tissue comparison and sometimes microdissection.81
Analytically, these methods show comparable performance
with an approximately 5% to 10% false-negative rate each.82
Mismatch repair IHC results can be falsely negative in the
setting of mutations (eg, in MLH1) that result in an
antigenically intact but functionally inactive protein, and
MSI can be falsely negative in the setting of intratumoral
heterogeneity and inadequate microdissection.83 The choice
of which method to use at a given institution usually
depends on factors such as cost, availability, and turnaround
time. Although using both tests will detect slightly more
cases than either test alone, the benefit is small. In the
setting of Lynch syndrome screening, a dMMR or MSI result
is typically followed by sequencing of specific MMR genes,
except in the case of MLH1 loss, when BRAF V600E and/or
promoter methylation testing can be used to confirm
somatic MMR deficiency.84
Although IHC-based assessment of MMR status has been
available for many years in the context of Lynch syndrome
screening, it is unclear what, if any, changes to the analytic
and interpretative aspects of testing will be required for the
application to cancer immunotherapy. Multiple IHC anti-
body clones for each of the 4 MMR proteins are available,
primarily as class 1 in vitro diagnostics, with 1 commercial
MMR panel having been FDA cleared for CRC as an aid in
the identification of probable Lynch syndrome.85,86 There are
currently no FDA-approved MMR/dMMR or MSI assays as
companion diagnostics for cancer immunotherapy, despite
the fact that the FDA has approved 2 cancer immunother-
apies for use in dMMR/MSI-H patient subsets.
The IHC determination of dMMR is based on identifying
loss of one or more MMR proteins, indicated by absent
nuclear staining in viable tumor nuclei in the presence of
unequivocal internal control cell staining. Although MMR
IHC staining patterns are usually consistent with the
biological function of the 4 MMR proteins, pathologists
should be aware of unusual staining patterns and diagnostic
pitfalls.85,87 Based on data in CRC, MMR IHC assessment in
biopsy samples may be preferable to surgical resection
specimens given that MMR protein staining loss has been
observed posttreatment.88–90 Although detection of BRAF
V600E via IHC can play a role in differentiating sporadic
CRC from Lynch syndrome,91 it does not have a role in the
identification of dMMR cancers potentially responsive to
cancer immunotherapy.
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
Figure 3. Prevalence of somatic mutations across human cancer types. Cancer subtypes ordered from lowest average mutation prevalence (left) to
highest (right). Note extensive variation of mutation prevalence within any single cancer subtype. Abbreviations: ALL, acute lymphocytic leukemia;
AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia. Reprinted by permission from Nature Publishing Group. Alexandrov LB et al.100
Signatures of mutational processes in human cancer. Nature. 2013;500(7463):415–421.
Microsatellite instability is traditionally assessed via PCR
analysis of specific microsatellite loci to determine if a
change in length due to insertion or deletion of DNA
repeats has occurred in the tumor as compared with normal
tissue. If the same number of DNA repeats is present in the
tumor and normal tissue, the result is considered microsat-
ellite stability. In contrast, if the number of DNA repeats
differs between tumor and normal, the result is considered
MSI. In 1998 the National Cancer Institute established
international criteria for MSI determination in CRC based
on 5 microsatellite markers, 2 mononucleotide markers
(BAT25 and BAT26), and 3 dinucleotide markers (D2S123,
D5S346, and D17S250).92 Subsequently, an alternative panel
containing 5 mononucleotide quasi-monomorphic markers
was proposed that demonstrated improved sensitivity
(95.8% versus 76.5%) and positive predictive value (88.5%
versus 65.0%) compared with the original National Cancer
Institute panel.93 Using either panel, a tumor is classified as
MSI-H if 30% or more of the repeats are unstable, MSI low
if less than 30% of repeats are unstable, and microsatellite
stable if no repeats are unstable.94
Microsatellite instability can also be assessed via next-
generation sequencing (NGS), and a limited number of
studies have shown good sensitivity and specificity using
NGS to assess MSI compared with traditional PCR
methods.95–97 The NGS assay must be designed to include
microsatellite regions, use bioinformatics algorithms that
can size these regions, and take into account the size
distribution for microsatellite regions in normal samples due
to polymerase slippage in vitro. Sensitivity of 96.4% to 100%
and specificity of 97.2% to 100% have been described for
both capture-based and PCR-based library preparation.98,99
Future cancer immunotherapy trials examining response
and survival rates in these discrepant cases will determine if
MSI-NGS provides any advantages over the traditional 5-
marker PCR panel. A clear advantage associated with MSI-
NGS is the ability to couple the analysis with broader
genomic analyses without adding an additional test or
consuming additional tumor material.
Arch Pathol Lab Med—Vol 144, June 2020
TMB (STATUS: EMERGING)
Background and Role in Cancer Immunotherapy
Tumor mutational burden is a measure of somatic cancer
mutation prevalence typically represented as the number of
mutations per megabase. Tumor mutational burden varies
significantly across and within human cancer types, with
melanoma, lung cancer, and bladder cancer having the
highest mutation prevalence (Figure 3).100 Although TMB is
related to dMMR and MSI, there is not complete overlap
between them; most dMMR/MSI-H tumors have high TMB,
but not all high-TMB tumors are dMMR/MSI-H.99 For these
reasons, TMB should be considered an independent
parameter and should not be used interchangeably with
dMMR or MSI.
The early realization that cancer immunotherapy response
rates were highest among tumor types with the highest
tumor mutation density led to several clinical outcome
studies to further investigate this correlation. The seminal
observation in 2014 by Snyder et al101 was that high
mutational load in melanoma correlated with sustained
clinical benefit from ipilimumab or tremelimumab-based
CTLA-4 inhibition. Importantly, the authors noted that this
correlation was imperfect, with some high-TMB patients not
benefiting from therapy. Several subsequent studies, in-
cluding those of Rizvi et al102 and Hellmann et al,103,104
demonstrated a similar association between TMB and
response to immunotherapy in lung cancer. The specific
immunotherapies, disease indications, and conclusions from
these studies are listed in Table 4.
Beyond melanoma and lung cancer, mutational load has
also been shown to be a predictor of response to checkpoint
blockade in a broad array of tumor types, suggesting that
high TMB could be used to identify optimally responsive
patient subsets across all cancers. Rosenberg et al105 found
mutational load to be an independent predictor of response
in metastatic urothelial carcinoma patients treated with
atezolizumab. In a retrospective analysis, Goodman et al106
also found that TMB was an independent predictor of
outcome in a study of 151 patients, multiple immunother-
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 715
 Table 4. Lung Cancer Studies Assessing Tumor Mutational Burden (TMB) and Cancer Immunotherapy Response
Study/Drug Findings Source, y
KEYNOTE-001/pembrolizumab Higher TMB was associated with improved objective response, durable Rizvi et al,102 2015
clinical benefit, and progression-free survival
CheckMate 227/nivolumab Progression-free survival was significantly longer versus chemotherapy among Hellmann et al,103 2018
þ ipilimumab patients with high TMB, irrespective of programmed death ligand-1
expression level
CheckMate 012/nivolumab TMB was the strongest feature associated with benefit in a multivariable Hellmann et al,104 2018
þ ipilimumab analysis
CheckMate 026/nivolumab Exploratory retrospective analysis suggests improvement in objective response Peters et al,188 2017
(first line) rate and progression-free survival compared with chemotherapy in patients
with high TMB

apies, and more than 20 diverse tumor types including
breast, cervical, ovarian, prostate, and renal cancers.
Evidence to date indicates that PD-L1 status and TMB are
independent biomarkers with largely additive ability to
predict benefit from immune checkpoint inhibition. Car-
bone et al107 did not observe an association between PD-L1
expression and TMB in an exploratory analysis within the
CheckMate 026 phase 3 study of nivolumab as first-line
treatment of advanced NSCLC patients, finding a higher
response rate in those patients with a high level of both
TMB and PD-L1 expression (75%) compared with patients
with a high level of either biomarker alone (32% for TMB,
34% for PD-L1) or patients with low levels of both
biomarkers (16%). Hellmann et al103,104 similarly found
TMB and PD-L1 expression to be independent biomarkers
in the CheckMate 227 trial103 and the CheckMate 012
trial,104 where NSCLC patients with positive PD-L1
expression (1%) and high TMB (.median) had signifi-
cantly improved response and progression-free survival
rates with combination nivolumab plus ipilimumab therapy
compared with patients whose tumors had only one or
neither variable.
TMB Testing
Several challenges need to be overcome before TMB can
be readily adopted into the routine clinical environment, the
most critical being testing access, technical/analytic burden,
and lack of standardized methods for TMB calculation and
reporting.
Most published studies demonstrating a correlation
between high TMB and clinical response to cancer
immunotherapies used whole-exome sequencing (WES), a
methodology not widely available or practical because of its
high cost and analytic burden. Targeted NGS is becoming
increasingly accessible across laboratory testing environ-
ments, and, when coupled with optimized bioinformatics
pipelines, can provide an accurate and practical method for
detecting the sequence changes used to calculate TMB. For
example, NGS-based comprehensive gene panels (CGPs)
sequence specific sets of genes (typically 300–600) related to
a focused disease or phenotype. These panels represent a
more routinely feasible option for assessing TMB in the
routine laboratory environment because of their lower cost,
faster turnaround time, and lower analytic burden compared
with WES. Several studies have found NGS-based TMB
assessment to be an accurate surrogate of TMB by WES.
Rizvi et al108 studied TMB assessment by targeted NGS
versus WES, finding a strong correlation between the 2
methods. Chalmers et al109 found that TMB from a 1.1-
megabase targeted CGP was strongly correlated (R2 ¼ 0.74)
716 Arch Pathol Lab Med—Vol 144, June 2020
with mutation burden determined by WES. Examining the
effect of various genomic parameters on quality, they found
that filtering out germline alterations and rare variants was
important in determining accurate TMB measurements.
The ability of NGS-based methods to accurately assess
TMB is dependent on the size of the target region
sequenced. Several groups have attempted to define the
minimum number of genes that can be used as the basis for
TMB, while remaining predictive of response to immuno-
therapy. Campesato et al110 assessed the ability of their
institutional CGP (641 genes) and the Foundation Medicine
CGP (315 genes) to estimate TMB, comparing the clinical
outcome prediction performance with WES-based TMB
from the melanoma and NSCLC checkpoint blockade trials
from Snyder et al101 and Rizvi et al,102 respectively. Tumor
mutational burden estimates from either of the CGPs were
significantly associated with durable clinical benefit in
NSCLC patients treated with PD-1 blockade. Predictive
accuracy of CGP-TMB for durable clinical benefit was not
statistically different from that of TMB from WES, with
similar area under the curve, sensitivity, and specificity
between the 2 CGPs and WES. Importantly, TMB predictive
accuracy was lost when smaller CGPs (,150 cancer genes)
were examined, causing the authors to recommend CGPs
with more than 300 cancer genes for TMB estimation. The
inability of smaller gene panels to accurately estimate TMB
is likely due to the sequenced region, typically in the range
of 25 kilobases, representing too small a portion of the
genome to accurately represent overall mutational burden.
Another challenge preventing widespread adoption of
TMB as a biomarker is the lack of standardization of TMB
calculation and reporting. For example, there is no standard
definition of high TMB, with some studies using various
absolute mutation thresholds102 and others using ratios of
mutations per megabase of DNA sequenced.111 The Friends
of Cancer Research, partnering with the National Cancer
Institute, the FDA, Johns Hopkins University, Memorial
Sloan Kettering Cancer Center, and multiple pharmaceutical
and diagnostic companies, hosted a TMB harmonization
working group meeting in May 2018 to begin the process of
addressing these challenges.112 Several opportunities for
TMB technical harmonization were provided, such as
agreement on analytic parameters for TMB calculation,
generation of a universal reference standard as an alignment
tool, and agreement on statistical approaches that will lead
to consistent TMB calculation and clinical interpretation.
Tumor mutational burden testing currently requires access
to tumor tissue, but noninvasive blood-based means of
estimating TMB are being explored. Gandara et al113
developed a novel blood-based assay to measure TMB
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
based on single-nucleotide variants from 394 genes from
cell-free DNA in patient plasma. Using blood samples from
the atezolizumab POPLAR (211 patients) and OAK (583
patients) trials in second-line NSCLC, the blood-based TMB
assay was predictive of clinical benefit independent of PD-
L1 expression. Prospective validation of this blood-based
assay as an alternative to tissue-based TMB would improve
the routine implementation of mutational burden assess-
ment in clinical practice.
POLE AND POLD1 MUTATIONS (STATUS: EMERGING)
Background and Biology
Polymerase d (POLD1 gene) and polymerase e (POLE
gene) are DNA polymerases involved in DNA replication
during the S phase of the cell cycle.114 These enzymes
additionally have DNA proofreading and repair functional-
ity via an exonuclease domain that allows excision and
replacement of incorrect bases and continuation of DNA
replication. Mutations of the exonuclease domain disrupt
the proofreading function and lead to an accumulation of
mutations elsewhere in the genome, often referred to the
ultramutator phenotype.114
POLE mutations associated with the ultramutator pheno-
type have been described in many different cancer types,
including about 3% of CRCs and 7% to 10% of endometrial
cancers.115–118 These tumors have thousands of mutations
across the genome, an inflamed tumor microenvironment,
and upregulated PD-L1, and are associated with a better
prognosis, similar to dMMR tumors. POLE mutations are
predominantly found in microsatellite-stable/pMMR tu-
mors, but one study identified them in MSI cases and
found that they account for some unexplained Lynch
syndrome phenotypes.119 POLD1 mutations appear to be
less prevalent than POLE mutations and occur more
commonly in dMMR tumors, but can also lead to the
ultramutator phenotype.120,121
Implications for Cancer Immunotherapy and Testing
Given the high mutation rate, inflamed tumor microen-
vironment, and upregulation of immune checkpoints
associated with POLE-mutated cancers, there is strong
scientific rationale to assess the efficacy of cancer immuno-
therapy in this setting. If effective, it would bring clinical
benefit to 1% to 3% of colon cancer patients not currently
addressed by cancer immunotherapy. Targeted NGS or
allele-specific PCR testing of POLE mutations could focus on
the 3 recurrent mutations (P286R/H/S, V411L, and S459F)
that represent 90% of the identified exonuclease domain
mutations.122 In addition to POLD1/POLE and MMR, there is
a strong rationale to evaluate deficiencies in other DNA
repair enzymes and mechanisms, such as O6-methylgua-
nine-DNA methyltransferase (MGMT), homologous recom-
bination, nucleotide excision repair, and base excision
repair, as potential biomarkers of immunotherapy response.
Several translational and clinical studies exploring these
DNA damage repair pathways in this context are currently
underway.123
CANCER NEOANTIGENS (STATUS: EARLY EMERGING)
The correlation of dMMR, MSI-H, TMB, and clinical
benefit from immune checkpoint blockade is thought to
represent a probabilistic relationship in which higher
mutational burden within a tumor increases the likelihood
of a neoantigen to which an effective T-cell immune
Arch Pathol Lab Med—Vol 144, June 2020
response can be generated.124 In this way, dMMR, MSI-H,
and TMB can all be thought of as surrogate measures of
immunogenic neoantigens, which may be the actual targets
of cytotoxic T cells in clinical cases of immunotherapy
response. In both melanoma and NSCLC patients, higher
neoantigen burden is associated with improved clinical
responses to immune checkpoint blockade therapy.125
Based on these hypotheses and early observations, there is
great interest in using tumor sequencing and bioinformatic
prediction algorithms to directly identify neoantigens that
can be used therapeutically in cancer vaccines126 and
diagnostically as more direct predictive biomarkers of cancer
immunotherapy efficacy. Experimental data exploring the
frequency, identity, and uniqueness of cancer neoantigens
have revealed new information that will guide the clinical
translation of this potentially important immunotherapy
biomarker.
Although human T cells have been shown to react to both
major histocompatibility complex class I–restricted and
major histocompatibility complex class II–restricted neo-
antigens in a large fraction of malignancies,127–130 only a
small percentage of tumor mutations actually lead to the
formation of neoantigens that are recognized by host T
cells.126,131–133 Additionally, it appears that immunoreactive
cancer neoantigens can arise from mutations in any gene
across the genome, and are not more common in driver
oncogenes related to tumorigenesis.126,129,134 Steven Rosen-
berg, MD (National Cancer Institute, Bethesda, Maryland),
pioneered a tandem minigene approach to identify the
precise neoantigens recognized by T cells from patients
treated with adoptive cell transfer.135,136 In melanoma,
neoantigens were identified in 29 of 31 patients (94%),
with each of the identified neoantigens being unique to the
autologous patient.135 In common gastrointestinal cancers,
neoantigen-reactive T cells were identified in 62 of 75
patients (83%), with 99% of the neoantigenic determinants
unique to each autologous patient.136 Immunogenic tumor
antigens associated with adoptive cell transfer complete
regressions have been identified in metastatic breast
cancer137 and human papillomavirus–associated metastatic
cervical cancer.138
Although this area is promising, several challenges would
need to be overcome before neoantigen prediction or
neoantigen load could become part of routine cancer
immunotherapy testing. These include lack of a complete
understanding of the underlying immunobiology, the low
frequency and apparent uniqueness of cancer neoantigens,
and a lack of suitable bioinformatics and laboratory methods
that can both provide accurate prediction and be imple-
mented into routine practice.
TIL ASSESSMENT AND MULTIPLEXED IMMUNE
PHENOTYPING (STATUS: EMERGING)
Pathologists have long recognized the presence of TILs in
diagnostic specimens, and many studies have explored their
role as biomarkers. In 2011, Mahmoud et al139 was the first
to identify TILs as a prognostic factor in breast cancer,
showing that tumor-infiltrating CD8þ lymphocytic density is
significantly associated with improved clinical outcome
independent of standard factors such as tumor grade,
lymph node stage, and HER2 status. In a study of stage I
to IV CRCs, Mlecnik et al140 found that an assessment of
CD8þ T cells in the tumor center and invasive margin was a
novel indicator of recurrence beyond TNM staging. Rakaee
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 717
et al141 developed and validated a hematoxylin-eosin–based
TIL scoring model, demonstrating that high stromal TIL
level is an independent measure of prognosis in stage I to III
NSCLC patients. Smyrk et al142 proposed TILs as a
screening criterion for selecting which CRC samples require
MSI testing, demonstrating a 93% sensitivity and 62%
specificity for MSI-H status with a TIL cutoff of 5. Assuming
a 12% MSI-H rate, they estimated that TIL assessment
could reduce the number of colon cancer samples referred to
MSI testing by more than 50% while still identifying 93% of
MSI-H cases.
Tumor-infiltrating lymphocytes indicate an inflamed
tumor microenvironment, and in the era of cancer
immunotherapy have been assessed as a predictive bio-
marker of response. Tumeh et al19 found a significantly
higher CD8þ T-cell density in the baseline biopsies of
melanoma patients responding to pembrolizumab com-
pared with the progression group, both at the invasive
margin and at the tumor center. However, there was no
CD8-positive cutoff value clearly separating responders
from nonresponders. Chen et al143 also found an increase
in the density of CD8þ, CD3þ, and CD45ROþ T cells in
pretreatment melanoma samples of CTLA-4 blockade
responders compared with nonresponders, but, similar to
Tumeh et al,19 could not establish a clear separation
between the 2 groups. However, their study also included
a separate analysis of anti–PD-1 treated patients, in which
on-treatment biopsies showed highly statistically significant,
largely nonoverlapping expression differences between
responders and nonresponders for CD8, CD4, and CD3 T-
cell subsets in addition to the immune checkpoint markers
PD-L1, PD-1, and LAG3.143 These changes were seen as
early as after 2 or 3 doses in responding patients. Also
observed in the early on-treatment biopsies was an
enrichment of CD8þ T cells in the tumor center versus the
tumor margin in responders compared with nonresponders,
suggesting therapy-induced tumor infiltration.
Beyond the assessment of TILs by hematoxylin-eosin
morphology and single-marker IHC, the development of
advanced in situ multiplexing methods has enabled more
comprehensive immunophenotyping of tumors and the host
microenvironment, including the ability to visualize the
colocalization of multiple immune-related markers.144 A
detailed overview of the currently available IHC multiplex-
ing methods and technologies is beyond the scope of this
article, but can be found in recently published reviews.145,146
The historic challenge of creating a multiplexed indirect IHC
assay that avoids multiple species of primary antibodies and
cross-reactivity between same-species primary antibodies
has largely been solved by elegant chemical and heat
deactivation reactions.145 Chromogenic multiplexing allows
light microscopy visualization, with novel methods such as
multiplexed immunohistochemical consecutive staining on
a single slide and reagents such as narrow-band tyramine-
conjugated dyes demonstrating the feasibility of medium- to
high-dimensional analysis.147,148 Fluorescence technologies
enable even higher-order multiplexing, with up to 61
individual analytes demonstrated with an iterative staining,
imaging, and chemical inactivation cycle approach.149 The
demonstration of fully automated fluorescence multiplexing
using tyramide-based covalent detection methods offers the
promise of these methods entering routine anatomic
pathology practice.150,151
Although initial studies using these novel techniques
focused on the characterization of specific intratumoral
718 Arch Pathol Lab Med—Vol 144, June 2020
immune subsets,147,152–154 some studies have begun to
correlate the phenotype and cellular interactions of immune
cells in the tumor microenvironment with immunotherapy
outcome data as a potentially more powerful predictor of
response. Giraldo et al155 found in the setting of Merkel cell
carcinoma that the number of pretreatment PD-1þ immune
cells in proximity to (within 15 lm of) a PD-L1þ immune or
tumor cell was associated with pembrolizumab response,
which was not the case with CD8/PD-L1 proximity.
Similarly, Johnson et al156 found that a PD-1/PD-L1
interaction score, but not PD-L1 alone, was associated with
anti–PD-1 response in metastatic melanoma.
Taken together, these studies demonstrate the potential of
TILs as an important cancer immunotherapy biomarker.
However, adoption of TIL assessment in any of its forms
into routine clinical practice will require robust methods and
tools for interpretation. Addressing the need for a stan-
dardized, reproducible approach to the histopathologic
assessment and quantification of TILs in routine practice,
Hendry et al,157,158 on behalf of the International Immuno-
oncology Biomarkers Working Group, have proposed a
hematoxylin-eosin–based methodology for assessing TILs in
solid tumors for clinical validity and utility assessment. In
addition, the International TILs Working Group has
published methodologic recommendations on the evalua-
tion of TILs in breast cancer, focusing on the stromal (versus
intratumoral) compartment.159 Beyond manual human
interpretation of TILs, computational image analysis–based
TIL scoring approaches have been developed, including
traditional object-oriented image-segmentation methods
and more advanced machine learning–based classification
algorithms that rely on extensive training sets but are able to
morphologically identify cellular subsets.160
TRANSCRIPTIONAL SIGNATURES OF IMMUNE
RESPONSIVENESS (STATUS: EMERGING)
RNA-based gene expression studies have been used to
identify specific transcriptional patterns and signatures in
tumors and the tumor microenvironment that may help
elucidate details of immune physiology underlying immu-
notherapy benefit. Summarized here are selected studies
exploring gene expression signatures that appear to play an
important role in the prediction of cancer immunotherapy
response.
Taube et al161,162 found that immune infiltrates at the
interface of PD-L1–positive melanoma cells demonstrate a
CD8þ T cell–T helper 1 cytokine mRNA signature charac-
terized by IFN-c expression, which was absent in PD-L1–
negative melanomas. They hypothesized that secretion of
the IFN-c cytokine not only triggers antitumor effects, but
simultaneously induces PD-L1 as part of an adaptive
negative-feedback immune regulatory mechanism. Since
this seminal discovery, the adaptive expression of PD-L1
has been observed in other tumor types, including Merkel
cell carcinoma,163 NSCLC,164 and breast cancer.165,166 Acti-
vation of the IFN-c signaling pathway is now recognized as
a necessary component of successful anticancer T-cell
immunity, an association reinforced by the discovery of
immunotherapy resistance mutations in Janus kinase 1
(JAK1) and Janus kinase 2 (JAK2) (see the Biomarkers of
Resistance to Cancer Immunotherapy section) that prevent
upregulation of IFN-c target genes.167
Ribas et al168 developed 2 gene signatures, IFN-c 10-gene
and expanded-immune 28-gene, both of which were
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
statistically associated with overall response rate and
progression-free survival in the context of melanoma treated
with pembrolizumab. In NSCLC, an 8-gene T-effector and
interferon-c gene signature was associated with improved
overall survival with atezolizumab in the POPLAR phase 2
trial.169 This result supports the hypothesis that preexisting
immunity is a predictor of benefit from checkpoint blockade.
In addition to having a possible role as a clinical
biomarker, gene expression signatures drive much of the
ongoing translational research into novel mechanisms of
cancer immunotherapy response and resistance that could
inform rational combination strategies.
BIOMARKERS OF RESISTANCE TO CANCER
IMMUNOTHERAPY (STATUS: EMERGING)
Although cancer immunotherapy delivers unprecedented
levels of durable clinical benefit to select patient subsets,
most patients initially treated with these therapies do not
respond (primary resistance) and of those who do respond,
some experience only short-lived benefit with eventual
tumor relapse (acquired or secondary resistance). This
section will briefly summarize some of the latest transla-
tional research into the mechanisms of immunotherapy
resistance.
One type of cancer immunotherapy resistance is immune
evasion resulting from tumor mutations that cause failures
in immune-mediated destruction and detection of cancer
cells. For example, Zaretsky et al167 identified JAK1 and
JAK2 mutations in 2 melanoma patients who initially
responded to pembrolizumab anti–PD-1 therapy but later
progressed. Mutations in JAK1/2 lead to abrogation of IFN-
c signaling and its antiproliferative effect on tumor cells.
Shin et al170 identified JAK 1/2 mutations as the cause of
primary resistance to anti–PD-1 therapy in dMMR colon
cancer and melanoma patients, demonstrating that these
mutations can cause both primary and secondary forms of
immunotherapy resistance.
Mutations causing impairment of the HLA class 1 antigen
processing and presentation machinery appears to be
another mechanism of resistance to cancer immunotherapy
agents. Gettinger et al171 identified homozygous loss of b2-
microglobulin (B2M), critical to proper functioning of the
HLA class 1 complex, in 1 of 14 lung cancer patients with
acquired resistance to immune checkpoint inhibition.
Zaretsky et al167 also identified a single patient with B2M
loss in their study of melanoma patients who progressed
after initially responding to immune checkpoint therapy.
Defects in several traditional cell signaling pathways have
also been correlated with immunotherapy resistance,
suggesting that many of these pathways can impact immune
regulation. Some pathways and genomic changes that have
been associated with negative immune regulation include b-
catenin activation (Spranger et al172), PTEN loss (Peng et
al173), and EGFR mutations and ALK rearrangements
(Gainor et al174). The apparent connectivity between these
pathways and immune activation/suppression provides a
strong rationale for combining targeted therapies and
immunotherapies.
Melanomas demonstrating primary resistance to check-
point inhibitors were found to display a specific transcrip-
tional signature referred to as innate anti–PD-1
resistance.175 Hugo et al175 found that this transcriptional
signature is characterized by increased expression of genes
involved in the regulation of extracellular matrix remodel-
Arch Pathol Lab Med—Vol 144, June 2020
ing, angiogenesis, wound healing, cell adhesion, and
mesenchymal transition. Activation of the innate anti–PD-
1 resistance signature was also demonstrated in lung
adenocarcinoma, colonic adenocarcinoma, renal clear cell
carcinoma, and pancreatic adenocarcinoma.175
Acknowledging that the outcome of cancer-immune
interactions is based on multiple independent parameters
that can each vary between patients, Blank et al176 have
proposed a ‘‘cancer immunogram’’ visualization framework
as a more comprehensive assessment of immune respon-
siveness and mechanisms of resistance. The immunogram
contains 7 initial parameter classes: tumor foreignness,
general immune status, immune cell infiltration, absence of
checkpoints, absence of soluble inhibitors, absence of
inhibitory tumor metabolism, and tumor sensitivity to
immune effectors. Resistance to immunotherapy is predict-
ed when any of the 7 parameters is unfavorable, and only
overcome with the appropriate targeting of the relevant
defect. Should this immunogram or a similar tool prove to
be valuable in the selection of responsive patients, its clinical
adoption will require the multifactorial integration of tumor
genomic, immunohistochemical, and peripheral blood data
by anatomic and molecular pathologists.
MICROBIOME (STATUS: EARLY EMERGING)
The human microbiome consists of the trillions of
microorganisms that colonize the skin, mucosal surfaces,
and gastrointestinal tract. The role of the microbiome and its
associated impact on human health and response to cancer
therapy has been proposed and explored in the past, but
until recently has been controversial. Recent conclusive data
supporting the role of the microbiome as a biomarker of
response to cancer immunotherapy have reignited scientific
interest in this field and triggered significant clinical
validation efforts.
Routy et al177 established a correlation between gut
microbiota and response to checkpoint inhibition, finding
a significant shortening of OS and progression-free survival
in NSCLC and urothelial carcinoma patients treated with
broad-spectrum antibiotics prior to anti–PD1/PD-L1 ther-
apy. Additionally, the authors found that gut microbiota
diversity, as measured by shotgun-sequencing gene count
or metagenomic species levels, correlated with clinical
response. They found enrichment of specific bacterial genera
in responders versus nonresponders, with Akkermansia
muciniphila having the most significant association with
favorable clinical outcome. They concluded that the gut
microbiome markedly influences PD-L1 blockade outcome
and hypothesized that certain bacteria such as A muciniphila
may reinforce intestinal barrier integrity, reduce systemic
inflammation, and improve immunosurveillance.
Matson et al178 used 16S ribosomal RNA gene amplicon
sequencing and species-specific quantitative PCR to study
stool samples from 42 metastatic melanoma patients
receiving immune checkpoint therapy, finding 8 bacterial
species more abundant in responders and 2 species more
abundant in nonresponders. Supporting the findings of
Routy et al,177 they identified A muciniphila in 4 patients, all
of whom were responders. They propose that multiple
specific bacteria may contribute to improved antitumor
immunity, and that the optimal biomarker may be a ratio of
beneficial bacteria that activate the immune system to
nonbeneficial bacteria that negatively regulate immune
responses. The authors conclude that the commensal
Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 719
microbiota may be a useful biomarker to predict response to
checkpoint blockade therapy, with patient responder-
associated bacteria having distinct local and systemic effects
on innate and adaptive immunity.
Gopalakrishnan et al179 prospectively collected micro-
biome samples from metastatic melanoma patients prior to
anti–PD-1 therapy. Within-sample gut microbiome diversity
was found to be significantly higher in responders versus
nonresponders, with greater diversity also correlating with
prolonged progression-free survival. Specific compositional
differences associated with anti–PD-1 therapy were identi-
fied, with Clostridiales order, Ruminococcaceae family, and
Faecalibacterium genus enriched in responders and Bacter-
oidales order enriched in nonresponders. Exploring poten-
tial mechanisms underlying these associations, the authors
found a statistically significant correlation between CD8þ T-
cell infiltrates in the tumor and abundance of Faecalibacte-
rium genus, Ruminococcaceae family, and Clostridiales
order in the gut, suggesting enhanced systemic and
antitumor responses mediated by increased antigen pre-
sentation and improved effector T-cell function. Finally,
germ-free mice transplanted with stool from responders to
anti–PD-1 therapy had higher CD8þ T-cell density and
improved response to immune checkpoint blockade com-
pared with those transplanted with stool from nonrespond-
ers, suggesting a causal link between a favorable gut
microbiome and immune checkpoint therapy.
Collectively, these studies provide compelling evidence
that the diversity and composition of the gut microbiome
could serve as a biomarker of cancer immunotherapy
response. Although additional validation via clinical trials
is necessary, the data also suggest that modulation of the
gut microbiome may have therapeutic potential for cancer
patients receiving immune checkpoint blockade.
CONCLUDING REMARKS
The field of cancer immunotherapy is still young and in
evolution, but has already transformed clinical oncology by
providing durable long-term survival benefit to previously
untreatable cancer patients. Because our ability to accurately
identify the minority of patients who currently derive benefit
from these therapies remains very limited, there is a critical
need to research, develop, and translate biomarkers
predictive of response.
From the landscape of cancer immunotherapy biomarkers
reviewed in this article, only PD-L1, MMR, and MSI testing
can be considered validated and routine because each is
associated with FDA-approved therapies. Tumor mutational
burden remains emerging, with issues of sensitivity/speci-
ficity and standardization yet to be resolved. Polymerase d
and e mutations are rare events but may help clarify which
pMMR patients derive benefit. Assessment of immunogenic
cancer neoantigens appears to be critical to understanding
the extent of tumor foreignness, but their detection and
identification are limited by current bioinformatic and
genomic technology. Tumor-infiltrating lymphocyte assess-
ment, especially enabled by multiplexed characterization of
immune cell subsets, is a promising emerging biomarker
that sits squarely in the domain of the anatomic pathologist.
Finally, transcriptional signatures of immune responsive-
ness, biomarkers of cancer immunotherapy resistance, and
the microbiome are all promising early emerging biomark-
ers of immunotherapy response that require additional
scientific, analytic, and clinical validation.
720 Arch Pathol Lab Med—Vol 144, June 2020
The biomarkers reviewed here represent only a small
proportion of the genomic, proteomic, and immunologic
parameters being explored in this dynamic and evolving
field. The breadth of exploration, in both the therapeutic and
diagnostic realms, is an indicator of our still nascent
understanding of the mechanisms underlying immune-
mediated cancer destruction. Adherence to the translational
medicine philosophy, whereby laboratory research leads to
novel therapeutic and diagnostic hypotheses that are then
clinically tested to inform subsequent scientific investiga-
tions, will continue to drive cancer immunotherapy pro-
gress, ultimately leading to better outcomes for patients.
As the field of cancer immunotherapy continues to
develop, pathologists bear an important responsibility to
lead and support studies that explore and validate novel
biomarkers. The current dynamism of this field represents
an opportunity for pathology to establish itself as the
immunotherapy biomarker center of excellence in clinical
medicine, ensuring the accurate, standardized, and timely
implementation of immunotherapy diagnostics through
education, training, and collaboration.
The authors would like to gratefully acknowledge Molly Hansen,
CT(ASCP), for her valuable assistance during the development of
this manuscript.
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