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Walk 2020
Walk 2020
Walk 2020
Context.—Cancer immunotherapy provides unprece- clinical trial data representing the current field of cancer
dented rates of durable clinical benefit to late-stage cancer immunotherapy.
patients across many tumor types, but there remains a Conclusions.—The cancer immunotherapy field, includ-
critical need for biomarkers to accurately predict clinical ing the use of biomarker testing to predict patient
response. Although some cancer immunotherapy tests are response, is still in evolution. PD-L1, mismatch repair,
associated with approved therapies and considered vali- and microsatellite instability testing are helping to guide
dated, other biomarkers are still emerging and at various the use of US Food and Drug Administration–approved
states of clinical and translational exploration. therapies, but there remains a need for better predictors of
Objective.—To provide pathologists with a current and response and resistance. Several categories of tumor and
practical update on the evolving field of cancer immuno-
patient characteristics underlying immune responsiveness
therapy testing. The scientific background, clinical data,
and testing methodology for the following cancer immu- are emerging and may represent the next generation of
notherapy biomarkers are reviewed: programmed death cancer immunotherapy predictive biomarkers. Pathologists
ligand-1 (PD-L1), mismatch repair, microsatellite instabil- have important roles and responsibilities as the field of
ity, tumor mutational burden, polymerase d and e cancer immunotherapy continues to develop, including
mutations, cancer neoantigens, tumor-infiltrating lympho- leadership of translational studies, exploration of novel
cytes, transcriptional signatures of immune responsiveness, biomarkers, and the accurate and timely implementation
cancer immunotherapy resistance biomarkers, and the of newly approved and validated companion diagnostics.
microbiome. (Arch Pathol Lab Med. 2020;144:706–724; doi: 10.5858/
Data Sources.—Selected scientific publications and arpa.2018-0584-CP)
approved by one or more global regulatory agencies across Clinical Oncology annual meeting) as ‘‘a test that aids in the
multiple cancer indications (Table 1).4 A recent systematic benefit-risk decision-making about the use of the thera-
review and meta-analysis by Khunger et al21 examined 41 peutic product, where the difference in benefit-risk is
PD-1/PD-L1 inhibitor clinical trials and found that PD-L1 clinically meaningful.’’25 In contrast, the FDA defines a
IHC positivity in tumor cells and immune cells was companion diagnostic as an ‘‘in vitro diagnostic device or an
predictive of favorable response across all tumor types. imaging tool that provides information that is essential for
Generally, PD-L1 expression is associated with a greater the safe and effective use of a corresponding therapeutic
likelihood of benefit from anti–PD-(L)1 therapy, but, in product.’’26
contrast to traditional companion diagnostic markers such
as HER2 or ALK, patients with PD-L1–negative tumors also PD-L1 Testing
benefit from these therapies up to 20% of the time, Four separate PD-L1 assays27–30 have been developed and
depending on the specific study, therapy, and indication.22–24 approved in association with the clinical development of the
In response to these data, the FDA created a novel different FDA-approved anti–PD-(L)1 therapies and cancer
diagnostic category termed complementary diagnostic, defined indications, leading to distinct therapy-indication-test com-
(in a draft definition given at the 2016 American Society of binations (Table 3). In addition, a fifth assay based on the
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
neous and/or temporal expression that was not detected, or usually caused by epigenetic silencing of MLH1 via
potentially not present, at the time of initial specimen promoter methylation and are frequently associated with
testing.45 Radiation therapy, chemotherapy, and small- BRAF V600E mutations.54,55
molecule kinase inhibitors have all been shown to induce Mismatch repair deficiency was first detected in colorectal
PD-L1 expression46–48 and therefore could represent bio- cancer (CRC) but can occur in many other tumor types56,57
logical causes of pretreatment versus posttreatment PD-L1 (Figure 2), with a prevalence of 4% across all adult solid
testing discrepancy. malignancies.58 Tumors with a significant frequency of
dMMR include endometrial, gastric, small intestinal, colo-
MMR AND MSI (STATUS: FDA-APPROVED THERAPIES rectal, cervical, prostate, bile duct, liver, and thyroid
REQUIRE TESTING) carcinomas; neuroendocrine tumors; and uterine sarco-
MMR Pathobiology mas.58 In one study, endometrial, gastric, and small
The DNA MMR system recognizes and corrects insertion, intestinal cancers were more likely to have MMR defects
deletion, and base pair mismatches that occur during DNA than colon cancer.59
replication.49 Deficiencies in MMR are primarily caused by Inherited deficiencies of MMR are the cause of hereditary
inactivation of one or more of the 4 main proteins: MLH1, nonpolyposis colon cancer, also known as Lynch syndrome,
MSH2, PMS2, and MSH6. Mismatch repair deficiency a genetic syndrome with a predisposition to cancer,
(dMMR) can be inherited or acquired (sporadic) and leads especially CRC and endometrial carcinoma (52%–82% and
to accumulation of 2 main types of mutations in the DNA: 25%–60% lifetime risk, respectively). Approximately 8%–
missense mutations throughout the genome and changes in 16% of dMMR CRC cases are associated with Lynch
the length of microsatellite regions.50,51 syndrome, with the remaining cases representing sporadic
Although inherited MMR defects can be caused by CRC.60–63 Identifying cases of Lynch syndrome is critical
inactivating mutations of any of the 4 MMR genes, MLH1 given the increased cancer risk not only for the patient (ie,
and MSH2 mutations account for approximately 90% of for other tumors), but also for the patient’s family members.
cases, with mutations in PMS2 and MSH6 accounting for Therefore, it is recommended that all cases of CRC and
most of the remaining cases.52,53 Sporadic MMR defects are endometrial carcinoma undergo screening for MMR de-
Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 713
fects.64–66 If dMMR is detected, further testing should be enabled activated T-cell immune response, are sufficient to
performed to determine if the defect is inherited or sporadic elicit antitumor immunity. Mismatch repair deficiency is
after the appropriate genetic counseling and consent is simply one mechanism that can create increased tumor
obtained. mutations, thereby increasing the probability of a neo-
antigen that can be processed and recognized by the
MSI Pathobiology adaptive immune system. For example, dMMR tumors
Microsatellites, also known as short tandem repeats or harbor a 10- to 100-fold higher rate of mutations compared
simple sequence repeats,67,68 are short repetitive DNA with pMMR tumors.74,75 High tumor mutational burden
sequences that occur commonly throughout the genome, (TMB) (see the TMB section) can be created via other defects
primarily within noncoding intergenic regions and introns. in the DNA editing machinery, such as loss of polymerase
During DNA replication, transient dissociation and subse- exonuclease function via POLE and POLD1 mutations (see
quent misaligned reassociation of the replicating DNA the POLE and POLD1 Mutations section).
strands can cause alterations in the length of these
microsatellites. These alterations are usually corrected by MMR and MSI Testing Methods
the MMR system, but in the setting of dMMR can remain There are 2 main methods of screening for MMR
uncorrected, resulting in MSI.69 Therefore, MSI is a genomic functional defects: IHC for the 4 MMR proteins MLH1,
consequence of dMMR, with the severity of instability MSH2, PMS2, and MSH6,76,77 and molecular PCR testing to
stratified by the number of affected microsatellite markers detect MSI.78–80 Each method has unique limitations and
into 3 groups: MSI high (MSI-H), MSI low, and microsat- diagnostic pitfalls, with IHC being dependent on fixation
ellite stable (see MMR and MSI Testing Methods below).69 conditions and accurate interpretation and MSI requiring
normal tissue comparison and sometimes microdissection.81
MMR, MSI, and Cancer Immunotherapy Analytically, these methods show comparable performance
Testing for dMMR and MSI has become a mandatory with an approximately 5% to 10% false-negative rate each.82
component of identifying patients most likely to respond to Mismatch repair IHC results can be falsely negative in the
immunotherapy targeting PD-(L)1 in certain indications. setting of mutations (eg, in MLH1) that result in an
The first clinical observation suggesting that MMR status antigenically intact but functionally inactive protein, and
predicted clinical response to anti–PD-1 blockade came MSI can be falsely negative in the setting of intratumoral
from a phase 2 study of the PD-1 checkpoint inhibitor heterogeneity and inadequate microdissection.83 The choice
pembrolizumab.70 Le et al70 hypothesized that the efficacy of which method to use at a given institution usually
rate of PD-1 blockade in CRC was associated with MMR depends on factors such as cost, availability, and turnaround
status and initiated a phase 2 clinical trial to evaluate time. Although using both tests will detect slightly more
pembrolizumab in patients whose tumors were dMMR or cases than either test alone, the benefit is small. In the
MMR proficient (pMMR). The results of this study showed a setting of Lynch syndrome screening, a dMMR or MSI result
dramatic difference in the objective response rate (ORR) of is typically followed by sequencing of specific MMR genes,
dMMR versus pMMR patients, with a 40% ORR in the except in the case of MLH1 loss, when BRAF V600E and/or
dMMR CRC patients versus 0% ORR in the pMMR CRC promoter methylation testing can be used to confirm
patients. Additionally, dMMR non-CRC patients, including somatic MMR deficiency.84
those with ampullary, endometrial, small bowel, and gastric Although IHC-based assessment of MMR status has been
cancers, also experienced a high rate of benefit with a 71% available for many years in the context of Lynch syndrome
ORR. screening, it is unclear what, if any, changes to the analytic
Currently, 2 cancer immunotherapy drugs are FDA and interpretative aspects of testing will be required for the
approved for dMMR tumors based on IHC or MSI application to cancer immunotherapy. Multiple IHC anti-
polymerase chain reaction (PCR) testing. In May 2017, the body clones for each of the 4 MMR proteins are available,
FDA granted accelerated approval to pembrolizumab for primarily as class 1 in vitro diagnostics, with 1 commercial
treatment of unresectable or metastatic dMMR or MSI-H MMR panel having been FDA cleared for CRC as an aid in
solid tumors that have progressed following prior treatment, the identification of probable Lynch syndrome.85,86 There are
the first cancer site–agnostic approval granted from the currently no FDA-approved MMR/dMMR or MSI assays as
FDA.71 The data for pembrolizumab were based on the companion diagnostics for cancer immunotherapy, despite
results from 5 single-arm multicenter trials (KEYNOTE-016, the fact that the FDA has approved 2 cancer immunother-
-164, -012, -028, and -158) of 149 total patients (90 CRC apies for use in dMMR/MSI-H patient subsets.
patients, 59 with 14 other cancer types) showing an ORR of The IHC determination of dMMR is based on identifying
40%, which was similar irrespective of tumor type.71 In these loss of one or more MMR proteins, indicated by absent
studies, determination of MSI-H or dMMR status for the nuclear staining in viable tumor nuclei in the presence of
majority of patients was prospectively performed via PCR unequivocal internal control cell staining. Although MMR
for MSI-H status or IHC for dMMR. The second FDA IHC staining patterns are usually consistent with the
approval related to MMR deficiency occurred when nivolu- biological function of the 4 MMR proteins, pathologists
mab was approved in July 2017 for CRC that has progressed should be aware of unusual staining patterns and diagnostic
following therapy, based on the CheckMate 142 study of 53 pitfalls.85,87 Based on data in CRC, MMR IHC assessment in
CRC patients showing an ORR of 32% in the overall biopsy samples may be preferable to surgical resection
population.72,73 specimens given that MMR protein staining loss has been
The association between dMMR response to cancer observed posttreatment.88–90 Although detection of BRAF
immunotherapy appears to be driven by the probabilistic V600E via IHC can play a role in differentiating sporadic
creation of nonself, cancer-specific antigens termed neo- CRC from Lynch syndrome,91 it does not have a role in the
antigens (see the Cancer Neoantigens section). These identification of dMMR cancers potentially responsive to
neoantigens, in the presence of a cancer immunotherapy– cancer immunotherapy.
714 Arch Pathol Lab Med—Vol 144, June 2020 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.
apies, and more than 20 diverse tumor types including with mutation burden determined by WES. Examining the
breast, cervical, ovarian, prostate, and renal cancers. effect of various genomic parameters on quality, they found
Evidence to date indicates that PD-L1 status and TMB are that filtering out germline alterations and rare variants was
independent biomarkers with largely additive ability to important in determining accurate TMB measurements.
predict benefit from immune checkpoint inhibition. Car- The ability of NGS-based methods to accurately assess
bone et al107 did not observe an association between PD-L1 TMB is dependent on the size of the target region
expression and TMB in an exploratory analysis within the sequenced. Several groups have attempted to define the
CheckMate 026 phase 3 study of nivolumab as first-line minimum number of genes that can be used as the basis for
treatment of advanced NSCLC patients, finding a higher TMB, while remaining predictive of response to immuno-
response rate in those patients with a high level of both therapy. Campesato et al110 assessed the ability of their
TMB and PD-L1 expression (75%) compared with patients institutional CGP (641 genes) and the Foundation Medicine
with a high level of either biomarker alone (32% for TMB, CGP (315 genes) to estimate TMB, comparing the clinical
34% for PD-L1) or patients with low levels of both outcome prediction performance with WES-based TMB
biomarkers (16%). Hellmann et al103,104 similarly found from the melanoma and NSCLC checkpoint blockade trials
TMB and PD-L1 expression to be independent biomarkers from Snyder et al101 and Rizvi et al,102 respectively. Tumor
in the CheckMate 227 trial103 and the CheckMate 012 mutational burden estimates from either of the CGPs were
trial,104 where NSCLC patients with positive PD-L1 significantly associated with durable clinical benefit in
expression (1%) and high TMB (.median) had signifi- NSCLC patients treated with PD-1 blockade. Predictive
cantly improved response and progression-free survival accuracy of CGP-TMB for durable clinical benefit was not
rates with combination nivolumab plus ipilimumab therapy statistically different from that of TMB from WES, with
compared with patients whose tumors had only one or similar area under the curve, sensitivity, and specificity
neither variable. between the 2 CGPs and WES. Importantly, TMB predictive
accuracy was lost when smaller CGPs (,150 cancer genes)
TMB Testing were examined, causing the authors to recommend CGPs
Several challenges need to be overcome before TMB can with more than 300 cancer genes for TMB estimation. The
be readily adopted into the routine clinical environment, the inability of smaller gene panels to accurately estimate TMB
most critical being testing access, technical/analytic burden, is likely due to the sequenced region, typically in the range
and lack of standardized methods for TMB calculation and of 25 kilobases, representing too small a portion of the
reporting. genome to accurately represent overall mutational burden.
Most published studies demonstrating a correlation Another challenge preventing widespread adoption of
between high TMB and clinical response to cancer TMB as a biomarker is the lack of standardization of TMB
immunotherapies used whole-exome sequencing (WES), a calculation and reporting. For example, there is no standard
methodology not widely available or practical because of its definition of high TMB, with some studies using various
high cost and analytic burden. Targeted NGS is becoming absolute mutation thresholds102 and others using ratios of
increasingly accessible across laboratory testing environ- mutations per megabase of DNA sequenced.111 The Friends
ments, and, when coupled with optimized bioinformatics of Cancer Research, partnering with the National Cancer
pipelines, can provide an accurate and practical method for Institute, the FDA, Johns Hopkins University, Memorial
detecting the sequence changes used to calculate TMB. For Sloan Kettering Cancer Center, and multiple pharmaceutical
example, NGS-based comprehensive gene panels (CGPs) and diagnostic companies, hosted a TMB harmonization
sequence specific sets of genes (typically 300–600) related to working group meeting in May 2018 to begin the process of
a focused disease or phenotype. These panels represent a addressing these challenges.112 Several opportunities for
more routinely feasible option for assessing TMB in the TMB technical harmonization were provided, such as
routine laboratory environment because of their lower cost, agreement on analytic parameters for TMB calculation,
faster turnaround time, and lower analytic burden compared generation of a universal reference standard as an alignment
with WES. Several studies have found NGS-based TMB tool, and agreement on statistical approaches that will lead
assessment to be an accurate surrogate of TMB by WES. to consistent TMB calculation and clinical interpretation.
Rizvi et al108 studied TMB assessment by targeted NGS Tumor mutational burden testing currently requires access
versus WES, finding a strong correlation between the 2 to tumor tissue, but noninvasive blood-based means of
methods. Chalmers et al109 found that TMB from a 1.1- estimating TMB are being explored. Gandara et al113
megabase targeted CGP was strongly correlated (R2 ¼ 0.74) developed a novel blood-based assay to measure TMB
716 Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
based on single-nucleotide variants from 394 genes from response can be generated.124 In this way, dMMR, MSI-H,
cell-free DNA in patient plasma. Using blood samples from and TMB can all be thought of as surrogate measures of
the atezolizumab POPLAR (211 patients) and OAK (583 immunogenic neoantigens, which may be the actual targets
patients) trials in second-line NSCLC, the blood-based TMB of cytotoxic T cells in clinical cases of immunotherapy
assay was predictive of clinical benefit independent of PD- response. In both melanoma and NSCLC patients, higher
L1 expression. Prospective validation of this blood-based neoantigen burden is associated with improved clinical
assay as an alternative to tissue-based TMB would improve responses to immune checkpoint blockade therapy.125
the routine implementation of mutational burden assess- Based on these hypotheses and early observations, there is
ment in clinical practice. great interest in using tumor sequencing and bioinformatic
prediction algorithms to directly identify neoantigens that
POLE AND POLD1 MUTATIONS (STATUS: EMERGING) can be used therapeutically in cancer vaccines126 and
Background and Biology diagnostically as more direct predictive biomarkers of cancer
immunotherapy efficacy. Experimental data exploring the
Polymerase d (POLD1 gene) and polymerase e (POLE frequency, identity, and uniqueness of cancer neoantigens
gene) are DNA polymerases involved in DNA replication have revealed new information that will guide the clinical
during the S phase of the cell cycle.114 These enzymes translation of this potentially important immunotherapy
additionally have DNA proofreading and repair functional-
biomarker.
ity via an exonuclease domain that allows excision and
Although human T cells have been shown to react to both
replacement of incorrect bases and continuation of DNA
major histocompatibility complex class I–restricted and
replication. Mutations of the exonuclease domain disrupt
major histocompatibility complex class II–restricted neo-
the proofreading function and lead to an accumulation of
antigens in a large fraction of malignancies,127–130 only a
mutations elsewhere in the genome, often referred to the
small percentage of tumor mutations actually lead to the
ultramutator phenotype.114
formation of neoantigens that are recognized by host T
POLE mutations associated with the ultramutator pheno-
cells.126,131–133 Additionally, it appears that immunoreactive
type have been described in many different cancer types,
cancer neoantigens can arise from mutations in any gene
including about 3% of CRCs and 7% to 10% of endometrial
cancers.115–118 These tumors have thousands of mutations across the genome, and are not more common in driver
across the genome, an inflamed tumor microenvironment, oncogenes related to tumorigenesis.126,129,134 Steven Rosen-
and upregulated PD-L1, and are associated with a better berg, MD (National Cancer Institute, Bethesda, Maryland),
prognosis, similar to dMMR tumors. POLE mutations are pioneered a tandem minigene approach to identify the
predominantly found in microsatellite-stable/pMMR tu- precise neoantigens recognized by T cells from patients
mors, but one study identified them in MSI cases and treated with adoptive cell transfer.135,136 In melanoma,
found that they account for some unexplained Lynch neoantigens were identified in 29 of 31 patients (94%),
syndrome phenotypes.119 POLD1 mutations appear to be with each of the identified neoantigens being unique to the
less prevalent than POLE mutations and occur more autologous patient.135 In common gastrointestinal cancers,
commonly in dMMR tumors, but can also lead to the neoantigen-reactive T cells were identified in 62 of 75
ultramutator phenotype.120,121 patients (83%), with 99% of the neoantigenic determinants
unique to each autologous patient.136 Immunogenic tumor
Implications for Cancer Immunotherapy and Testing antigens associated with adoptive cell transfer complete
Given the high mutation rate, inflamed tumor microen- regressions have been identified in metastatic breast
vironment, and upregulation of immune checkpoints cancer137 and human papillomavirus–associated metastatic
associated with POLE-mutated cancers, there is strong cervical cancer.138
scientific rationale to assess the efficacy of cancer immuno- Although this area is promising, several challenges would
therapy in this setting. If effective, it would bring clinical need to be overcome before neoantigen prediction or
benefit to 1% to 3% of colon cancer patients not currently neoantigen load could become part of routine cancer
addressed by cancer immunotherapy. Targeted NGS or immunotherapy testing. These include lack of a complete
allele-specific PCR testing of POLE mutations could focus on understanding of the underlying immunobiology, the low
the 3 recurrent mutations (P286R/H/S, V411L, and S459F) frequency and apparent uniqueness of cancer neoantigens,
that represent 90% of the identified exonuclease domain and a lack of suitable bioinformatics and laboratory methods
mutations.122 In addition to POLD1/POLE and MMR, there is that can both provide accurate prediction and be imple-
a strong rationale to evaluate deficiencies in other DNA mented into routine practice.
repair enzymes and mechanisms, such as O6-methylgua-
nine-DNA methyltransferase (MGMT), homologous recom- TIL ASSESSMENT AND MULTIPLEXED IMMUNE
bination, nucleotide excision repair, and base excision PHENOTYPING (STATUS: EMERGING)
repair, as potential biomarkers of immunotherapy response. Pathologists have long recognized the presence of TILs in
Several translational and clinical studies exploring these diagnostic specimens, and many studies have explored their
DNA damage repair pathways in this context are currently role as biomarkers. In 2011, Mahmoud et al139 was the first
underway.123 to identify TILs as a prognostic factor in breast cancer,
showing that tumor-infiltrating CD8þ lymphocytic density is
CANCER NEOANTIGENS (STATUS: EARLY EMERGING) significantly associated with improved clinical outcome
The correlation of dMMR, MSI-H, TMB, and clinical independent of standard factors such as tumor grade,
benefit from immune checkpoint blockade is thought to lymph node stage, and HER2 status. In a study of stage I
represent a probabilistic relationship in which higher to IV CRCs, Mlecnik et al140 found that an assessment of
mutational burden within a tumor increases the likelihood CD8þ T cells in the tumor center and invasive margin was a
of a neoantigen to which an effective T-cell immune novel indicator of recurrence beyond TNM staging. Rakaee
Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 717
et al141 developed and validated a hematoxylin-eosin–based immune subsets,147,152–154 some studies have begun to
TIL scoring model, demonstrating that high stromal TIL correlate the phenotype and cellular interactions of immune
level is an independent measure of prognosis in stage I to III cells in the tumor microenvironment with immunotherapy
NSCLC patients. Smyrk et al142 proposed TILs as a outcome data as a potentially more powerful predictor of
screening criterion for selecting which CRC samples require response. Giraldo et al155 found in the setting of Merkel cell
MSI testing, demonstrating a 93% sensitivity and 62% carcinoma that the number of pretreatment PD-1þ immune
specificity for MSI-H status with a TIL cutoff of 5. Assuming cells in proximity to (within 15 lm of) a PD-L1þ immune or
a 12% MSI-H rate, they estimated that TIL assessment tumor cell was associated with pembrolizumab response,
could reduce the number of colon cancer samples referred to which was not the case with CD8/PD-L1 proximity.
MSI testing by more than 50% while still identifying 93% of Similarly, Johnson et al156 found that a PD-1/PD-L1
MSI-H cases. interaction score, but not PD-L1 alone, was associated with
Tumor-infiltrating lymphocytes indicate an inflamed anti–PD-1 response in metastatic melanoma.
tumor microenvironment, and in the era of cancer Taken together, these studies demonstrate the potential of
immunotherapy have been assessed as a predictive bio- TILs as an important cancer immunotherapy biomarker.
marker of response. Tumeh et al19 found a significantly However, adoption of TIL assessment in any of its forms
higher CD8þ T-cell density in the baseline biopsies of into routine clinical practice will require robust methods and
melanoma patients responding to pembrolizumab com- tools for interpretation. Addressing the need for a stan-
pared with the progression group, both at the invasive dardized, reproducible approach to the histopathologic
margin and at the tumor center. However, there was no assessment and quantification of TILs in routine practice,
CD8-positive cutoff value clearly separating responders Hendry et al,157,158 on behalf of the International Immuno-
from nonresponders. Chen et al143 also found an increase oncology Biomarkers Working Group, have proposed a
in the density of CD8þ, CD3þ, and CD45ROþ T cells in hematoxylin-eosin–based methodology for assessing TILs in
pretreatment melanoma samples of CTLA-4 blockade solid tumors for clinical validity and utility assessment. In
responders compared with nonresponders, but, similar to addition, the International TILs Working Group has
Tumeh et al,19 could not establish a clear separation published methodologic recommendations on the evalua-
between the 2 groups. However, their study also included tion of TILs in breast cancer, focusing on the stromal (versus
a separate analysis of anti–PD-1 treated patients, in which intratumoral) compartment.159 Beyond manual human
on-treatment biopsies showed highly statistically significant, interpretation of TILs, computational image analysis–based
largely nonoverlapping expression differences between TIL scoring approaches have been developed, including
responders and nonresponders for CD8, CD4, and CD3 T- traditional object-oriented image-segmentation methods
cell subsets in addition to the immune checkpoint markers and more advanced machine learning–based classification
PD-L1, PD-1, and LAG3.143 These changes were seen as algorithms that rely on extensive training sets but are able to
early as after 2 or 3 doses in responding patients. Also morphologically identify cellular subsets.160
observed in the early on-treatment biopsies was an
enrichment of CD8þ T cells in the tumor center versus the TRANSCRIPTIONAL SIGNATURES OF IMMUNE
tumor margin in responders compared with nonresponders, RESPONSIVENESS (STATUS: EMERGING)
suggesting therapy-induced tumor infiltration.
Beyond the assessment of TILs by hematoxylin-eosin RNA-based gene expression studies have been used to
morphology and single-marker IHC, the development of identify specific transcriptional patterns and signatures in
advanced in situ multiplexing methods has enabled more tumors and the tumor microenvironment that may help
comprehensive immunophenotyping of tumors and the host elucidate details of immune physiology underlying immu-
microenvironment, including the ability to visualize the notherapy benefit. Summarized here are selected studies
colocalization of multiple immune-related markers.144 A exploring gene expression signatures that appear to play an
detailed overview of the currently available IHC multiplex- important role in the prediction of cancer immunotherapy
ing methods and technologies is beyond the scope of this response.
article, but can be found in recently published reviews.145,146 Taube et al161,162 found that immune infiltrates at the
The historic challenge of creating a multiplexed indirect IHC interface of PD-L1–positive melanoma cells demonstrate a
assay that avoids multiple species of primary antibodies and CD8þ T cell–T helper 1 cytokine mRNA signature charac-
cross-reactivity between same-species primary antibodies terized by IFN-c expression, which was absent in PD-L1–
has largely been solved by elegant chemical and heat negative melanomas. They hypothesized that secretion of
deactivation reactions.145 Chromogenic multiplexing allows the IFN-c cytokine not only triggers antitumor effects, but
light microscopy visualization, with novel methods such as simultaneously induces PD-L1 as part of an adaptive
multiplexed immunohistochemical consecutive staining on negative-feedback immune regulatory mechanism. Since
a single slide and reagents such as narrow-band tyramine- this seminal discovery, the adaptive expression of PD-L1
conjugated dyes demonstrating the feasibility of medium- to has been observed in other tumor types, including Merkel
high-dimensional analysis.147,148 Fluorescence technologies cell carcinoma,163 NSCLC,164 and breast cancer.165,166 Acti-
enable even higher-order multiplexing, with up to 61 vation of the IFN-c signaling pathway is now recognized as
individual analytes demonstrated with an iterative staining, a necessary component of successful anticancer T-cell
imaging, and chemical inactivation cycle approach.149 The immunity, an association reinforced by the discovery of
demonstration of fully automated fluorescence multiplexing immunotherapy resistance mutations in Janus kinase 1
using tyramide-based covalent detection methods offers the (JAK1) and Janus kinase 2 (JAK2) (see the Biomarkers of
promise of these methods entering routine anatomic Resistance to Cancer Immunotherapy section) that prevent
pathology practice.150,151 upregulation of IFN-c target genes.167
Although initial studies using these novel techniques Ribas et al168 developed 2 gene signatures, IFN-c 10-gene
focused on the characterization of specific intratumoral and expanded-immune 28-gene, both of which were
718 Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al
statistically associated with overall response rate and ing, angiogenesis, wound healing, cell adhesion, and
progression-free survival in the context of melanoma treated mesenchymal transition. Activation of the innate anti–PD-
with pembrolizumab. In NSCLC, an 8-gene T-effector and 1 resistance signature was also demonstrated in lung
interferon-c gene signature was associated with improved adenocarcinoma, colonic adenocarcinoma, renal clear cell
overall survival with atezolizumab in the POPLAR phase 2 carcinoma, and pancreatic adenocarcinoma.175
trial.169 This result supports the hypothesis that preexisting Acknowledging that the outcome of cancer-immune
immunity is a predictor of benefit from checkpoint blockade. interactions is based on multiple independent parameters
In addition to having a possible role as a clinical that can each vary between patients, Blank et al176 have
biomarker, gene expression signatures drive much of the proposed a ‘‘cancer immunogram’’ visualization framework
ongoing translational research into novel mechanisms of as a more comprehensive assessment of immune respon-
cancer immunotherapy response and resistance that could siveness and mechanisms of resistance. The immunogram
inform rational combination strategies. contains 7 initial parameter classes: tumor foreignness,
general immune status, immune cell infiltration, absence of
BIOMARKERS OF RESISTANCE TO CANCER checkpoints, absence of soluble inhibitors, absence of
IMMUNOTHERAPY (STATUS: EMERGING) inhibitory tumor metabolism, and tumor sensitivity to
Although cancer immunotherapy delivers unprecedented immune effectors. Resistance to immunotherapy is predict-
levels of durable clinical benefit to select patient subsets, ed when any of the 7 parameters is unfavorable, and only
most patients initially treated with these therapies do not overcome with the appropriate targeting of the relevant
respond (primary resistance) and of those who do respond, defect. Should this immunogram or a similar tool prove to
some experience only short-lived benefit with eventual be valuable in the selection of responsive patients, its clinical
tumor relapse (acquired or secondary resistance). This adoption will require the multifactorial integration of tumor
section will briefly summarize some of the latest transla- genomic, immunohistochemical, and peripheral blood data
tional research into the mechanisms of immunotherapy by anatomic and molecular pathologists.
resistance.
One type of cancer immunotherapy resistance is immune MICROBIOME (STATUS: EARLY EMERGING)
evasion resulting from tumor mutations that cause failures The human microbiome consists of the trillions of
in immune-mediated destruction and detection of cancer microorganisms that colonize the skin, mucosal surfaces,
cells. For example, Zaretsky et al167 identified JAK1 and and gastrointestinal tract. The role of the microbiome and its
JAK2 mutations in 2 melanoma patients who initially associated impact on human health and response to cancer
responded to pembrolizumab anti–PD-1 therapy but later therapy has been proposed and explored in the past, but
progressed. Mutations in JAK1/2 lead to abrogation of IFN- until recently has been controversial. Recent conclusive data
c signaling and its antiproliferative effect on tumor cells. supporting the role of the microbiome as a biomarker of
Shin et al170 identified JAK 1/2 mutations as the cause of response to cancer immunotherapy have reignited scientific
primary resistance to anti–PD-1 therapy in dMMR colon interest in this field and triggered significant clinical
cancer and melanoma patients, demonstrating that these validation efforts.
mutations can cause both primary and secondary forms of Routy et al177 established a correlation between gut
immunotherapy resistance. microbiota and response to checkpoint inhibition, finding
Mutations causing impairment of the HLA class 1 antigen a significant shortening of OS and progression-free survival
processing and presentation machinery appears to be in NSCLC and urothelial carcinoma patients treated with
another mechanism of resistance to cancer immunotherapy broad-spectrum antibiotics prior to anti–PD1/PD-L1 ther-
agents. Gettinger et al171 identified homozygous loss of b2- apy. Additionally, the authors found that gut microbiota
microglobulin (B2M), critical to proper functioning of the diversity, as measured by shotgun-sequencing gene count
HLA class 1 complex, in 1 of 14 lung cancer patients with or metagenomic species levels, correlated with clinical
acquired resistance to immune checkpoint inhibition. response. They found enrichment of specific bacterial genera
Zaretsky et al167 also identified a single patient with B2M in responders versus nonresponders, with Akkermansia
loss in their study of melanoma patients who progressed muciniphila having the most significant association with
after initially responding to immune checkpoint therapy. favorable clinical outcome. They concluded that the gut
Defects in several traditional cell signaling pathways have microbiome markedly influences PD-L1 blockade outcome
also been correlated with immunotherapy resistance, and hypothesized that certain bacteria such as A muciniphila
suggesting that many of these pathways can impact immune may reinforce intestinal barrier integrity, reduce systemic
regulation. Some pathways and genomic changes that have inflammation, and improve immunosurveillance.
been associated with negative immune regulation include b- Matson et al178 used 16S ribosomal RNA gene amplicon
catenin activation (Spranger et al172), PTEN loss (Peng et sequencing and species-specific quantitative PCR to study
al173), and EGFR mutations and ALK rearrangements stool samples from 42 metastatic melanoma patients
(Gainor et al174). The apparent connectivity between these receiving immune checkpoint therapy, finding 8 bacterial
pathways and immune activation/suppression provides a species more abundant in responders and 2 species more
strong rationale for combining targeted therapies and abundant in nonresponders. Supporting the findings of
immunotherapies. Routy et al,177 they identified A muciniphila in 4 patients, all
Melanomas demonstrating primary resistance to check- of whom were responders. They propose that multiple
point inhibitors were found to display a specific transcrip- specific bacteria may contribute to improved antitumor
tional signature referred to as innate anti–PD-1 immunity, and that the optimal biomarker may be a ratio of
resistance.175 Hugo et al175 found that this transcriptional beneficial bacteria that activate the immune system to
signature is characterized by increased expression of genes nonbeneficial bacteria that negatively regulate immune
involved in the regulation of extracellular matrix remodel- responses. The authors conclude that the commensal
Arch Pathol Lab Med—Vol 144, June 2020 Cancer Immunotherapy Biomarker Testing Landscape—Walk et al 719
microbiota may be a useful biomarker to predict response to The biomarkers reviewed here represent only a small
checkpoint blockade therapy, with patient responder- proportion of the genomic, proteomic, and immunologic
associated bacteria having distinct local and systemic effects parameters being explored in this dynamic and evolving
on innate and adaptive immunity. field. The breadth of exploration, in both the therapeutic and
Gopalakrishnan et al179 prospectively collected micro- diagnostic realms, is an indicator of our still nascent
biome samples from metastatic melanoma patients prior to understanding of the mechanisms underlying immune-
anti–PD-1 therapy. Within-sample gut microbiome diversity mediated cancer destruction. Adherence to the translational
was found to be significantly higher in responders versus medicine philosophy, whereby laboratory research leads to
nonresponders, with greater diversity also correlating with novel therapeutic and diagnostic hypotheses that are then
prolonged progression-free survival. Specific compositional clinically tested to inform subsequent scientific investiga-
differences associated with anti–PD-1 therapy were identi- tions, will continue to drive cancer immunotherapy pro-
fied, with Clostridiales order, Ruminococcaceae family, and gress, ultimately leading to better outcomes for patients.
Faecalibacterium genus enriched in responders and Bacter- As the field of cancer immunotherapy continues to
oidales order enriched in nonresponders. Exploring poten- develop, pathologists bear an important responsibility to
tial mechanisms underlying these associations, the authors lead and support studies that explore and validate novel
found a statistically significant correlation between CD8þ T- biomarkers. The current dynamism of this field represents
cell infiltrates in the tumor and abundance of Faecalibacte- an opportunity for pathology to establish itself as the
rium genus, Ruminococcaceae family, and Clostridiales immunotherapy biomarker center of excellence in clinical
order in the gut, suggesting enhanced systemic and medicine, ensuring the accurate, standardized, and timely
antitumor responses mediated by increased antigen pre- implementation of immunotherapy diagnostics through
sentation and improved effector T-cell function. Finally, education, training, and collaboration.
germ-free mice transplanted with stool from responders to
anti–PD-1 therapy had higher CD8þ T-cell density and The authors would like to gratefully acknowledge Molly Hansen,
improved response to immune checkpoint blockade com- CT(ASCP), for her valuable assistance during the development of
pared with those transplanted with stool from nonrespond- this manuscript.
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