Assessing tumor infiltrating lymphocytes in solid tumors: a
practical review for pathologists and proposal for a standardized
method from the International Immuno-Oncology Biomarkers
Working Group:
Part 1: Assessing the host immune response, TILs in invasive breast carcinoma and ductal
carcinoma in situ, metastatic tumor deposits and areas for further research
A full list of authors and affiliations appears at the end of the article.
Abstract
Assessment of tumor infiltrating lymphocytes (TILs) in histopathological specimens can provide
important prognostic information in diverse solid tumor types, and may also be of value in
predicting response to treatments. However, implementation as a routine clinical biomarker has
not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of
immunotherapy become a clinical reality, the need for widely applicable, accessible and reliable
immuno-oncology biomarkers is clear. In Part 1 of this review we briefly discuss the host immune
response to tumors and different approaches to TIL assessment. We propose a standardized
methodology to assess TILs in solid tumors on H&E sections, in both primary and metastatic
settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for
TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL
assessment in different solid tumor types follows in Part 2. The method we propose is
reproducible, affordable, easily applied, and has demonstrated prognostic and predictive
significance in invasive breast carcinoma. This standardized methodology may be used as a
reference against which other methods are compared, and should be evaluated for clinical validity
and utility. Standardization of TIL assessment will help to improve consistency and reproducibility
in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly
evaluate the utility of TILs assessment in this era of immunotherapy.
Keywords
Lymphocytes; tumor-infiltrating; Biomarkers; Cancer; Immunotherapy; Pathology
Corresponding author: Dr. Roberto Salgado, Department of Pathology, Oosterveldlaan 24, 2610 Wilrijk, Belgium,
roberto@salgado.be, Tel: ++32 497 89 12 18. Prof. Stephen Fox, Department of Pathology, Peter MacCallum Cancer Centre, 305
Grattan St, Melbourne, Victoria 3000, Australia, stephen.fox@petermac.org, Tel: +61 3 8559 5464.
The authors have no conflicts of interest to disclose.
HHS Public Access
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Adv Anat Pathol
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Published in final edited form as:
Adv Anat Pathol
. 2017 September ; 24(5): 235–251. doi:10.1097/PAP.0000000000000162.
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Introduction
Pathologists have long recognized the stroma, immune infiltrate, nerves and vasculature as
integral parts of the tumor microenvironment, which often provide important information
regarding tumor behavior, prognosis and response to treatment. It is well established that
tumors are antigenic and can induce an immune response, due in part to altered protein
products that may be recognized as foreign by the host immune system [1,2]. A growing
body of research has shown that the extent and composition of the host immune response to
the tumor has prognostic and predictive significance in many solid malignancies (reviewed
in [3]). The assessment of immune infiltrate in tumors, most commonly referred to as tumor
infiltrating lymphocytes (TILs), is also gaining importance in the current quest for optimal
biomarkers to select patients with the highest likelihood of responding to immunotherapeutic
agents. Therefore, TIL assessment has been proposed as a biomarker for inclusion in routine
histopathological reporting [4,5]. Current TIL scoring systems used in research and
proposed for different tumor types vary widely in detail, scope, accuracy, and time and
resource requirements.
Development of prognostic and predictive biomarkers in oncology requires robust
assessment of the test’s analytical validity, clinical validity and clinical utility [6,7].
Evidence is accumulating to support the use of TILs scoring as a prognostic biomarker in
various solid tumors and evidence for the predictive benefit of TILs is being investigated at
present. Different methods of assessing TILs will have different pre-analytical, analytical
and post-analytical challenges. For example, semi-quantitative H&E based scores may suffer
from low precision and poor inter-observer reproducibility if no clear guidance exists, while
digital quantification of IHC stained sections may produce different results due to inaccurate
measurement of the test variable without controlled calibration. Testing of the clinical
validity of biomarkers involves determining the extent to which the biomarker predicts the
clinical outcome of interest, that is, patient prognosis or response to treatment [7].
Assessment of the clinical validity of TILs scoring requires a standardized, reproducible
method, which can be validated preferably in several independent populations. Many
biomarker studies are observational, retrospective studies in which the study population is
selected solely by the availability of samples [8,9]. While prospective controlled studies
designed to test biomarkers are rare and unlikely to be performed on a large scale,
prospective-retrospective studies may offer a comparable level of evidence [8]. These
prospective-retrospective studies involve use of samples collected during a prospective
randomized clinical trial, and allow high quality evaluation of the biomarker of interest
provided the study design meets certain criteria and results can be replicated in an
independent population [8]. Guidelines for the reporting of biomarker studies are available
[9,10] and should be considered when evaluating the TILs literature.
In part 1 of this review, we aim to briefly describe the host immune response to tumors and
approaches used to assess this in the current context of immunotherapy. We propose a
standardized methodology for TIL assessment in solid tumors, based on the International
Immuno-Oncology Biomarkers Working Group guidelines for TIL assessment in invasive
breast carcinoma, which may be adapted to different tumor types. We then discuss the
literature and our experiences in the areas of invasive breast cancer, ductal carcinoma in situ
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(DCIS) and metastatic tumor deposits, then conclude with a discussion of open questions
and areas for further research. In part 2 of this review, the literature surrounding methods of
TIL assessment and the prognostic and potentially predictive significance of TILs in
different solid tumors is discussed, including carcinomas of the lung, colon, upper
gastrointestinal tract, head and neck, genitourinary tract, and gynecological organs, as well
as mesothelioma, melanoma and primary brain tumors. Ways in which the proposed
methodology can be adapted to different tumors are suggested, based on available evidence
and expert opinion. Standardization of TIL assessment will allow direct comparison of
different studies, highlight areas for further research, and form the basis of TIL assessment
in routine histological practice.
The host immune response
Altered protein products caused by the genetic mutations in cancer cells can function as
neoantigens, eliciting an immune response against a perceived “foreign” cell [2]. In addition,
the inflammatory, hypoxic and often necrotic microenvironment of tumors sends
concomitant danger signals to the host immune system [11]. Infiltrating immune cells can
function to control tumor growth and progression, but can also help to create an
immunosuppressive environment in which the tumor can thrive [12]. CD8
+
cytotoxic T cells,
T-helper 1 (Th1) cells producing interferon-γ, and natural killer cells are generally
associated with favorable anti-tumor immune responses, along with macrophages polarized
to an M1 phenotype and dendritic cells showing a DC1 phenotype. Immunosuppressive
effects are seen with Th2 cells, M2 macrophages, DC2 dendritic cells, myeloid derived
suppressor cells and FOXP3
+
regulatory T (T
reg
) cells producing IL-10 and TGFβ. B cells
and plasma cells can also adopt either effector or regulatory phenotypes, and hence can carry
positive or negative anti-tumor associations depending on contextual factors. This balance of
the cellular constituents of the immune response is illustrated in Figure 1. The presence of
tertiary lymphoid structures, aggregates which recapitulate the components and architecture
of a lymph node, in the tumor microenvironment is correlated with better prognosis in
different types of solid tumors (reviewed in [13]). The exact composition of the immune
infiltrate can vary widely within and between tumors and clearly modulate the effectiveness
of the anti-tumor response.
In research settings, many different methods are being used to investigate the host immune
response to tumors. Many clinical studies have found significant results using an assessment
of H&E stained sections by trained pathologists, with qualitative or semi-quantitative
scoring systems which vary according to tumor type [14–16]. Immunohistochemistry (IHC)
allows definition of the majority of immune cell subsets that can be refined by combinations
of markers, including CD8
+
cytotoxic T cells, CD4
+
T helper cells, FOXP3
+
T
regs
, B cells,
macrophages and dendritic cells. Other cell types such as myeloid derived suppressor cells
require multiple cell surface markers for definition and are challenging to identify on serial
IHC sections. Digital image analysis has been validated in multiple studies and can provide
accurate quantitation of immune cell infiltrates in IHC stained sections [17,18]. Multiplexed
fluorescent immunohistochemistry with multispectral imaging is a recent development that
allows in-situ identification of different immune cell subsets on the same section, providing
quantitative information on the distribution and composition of the immune infiltrate on
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formalin fixed, paraffin embedded (FFPE) tissue [19–21]. This technology requires a
significant investment in initial optimization, has complex data analysis and storage
requirements, and is yet to become routine. Recently, a novel approach to multiplexed IHC
was described utilizing NanoString® nCounter® fluorescent barcodes to identify bound
antibodies, allowing quantitation of multiple proteins in situ on an FFPE slide [22]. FFPE
tissue can also be used for matrix assisted laser desorption/ionization-imaging mass
spectrometry (MALDI-IMS), a proteomic technique that can identify hundreds proteins in
situ without the need for specific antibodies [23,24]. These exciting new technologies,
termed “molecular histology” combine the spatial and architectural information from
traditional histology approaches with detailed molecular profiling, which is likely to be
particularly relevant in describing the immune microenvironment of tumors.
Flow cytometry is a common approach to immune cell profiling and has many benefits
including the characterization of immune cell subsets by multiple markers, quantitative data
acquisition, wide availability and the ability to examine small subpopulations of interest
[25]. However, fresh tissue is required and no information is provided on the distribution or
organization of the immune infiltrate or relationship to other microenvironmental structures.
Nevertheless, recent study of TILs in invasive breast carcinoma found a significant positive
correlation between fresh tumor tissue analyzed by flow cytometry and IHC stained sections
scored by a pathologist [26]. Messenger RNA (mRNA) profiling of tumor tissue can detect
“immune gene signatures”, using the level of expression of immune related genes to
describe the composition and functional status of the immune infiltrate [27]. Again, no
information is provided on the distribution of the infiltrate and this resource intensive
technology is currently largely restricted to a research setting. These more complex methods
are by their nature more difficult to implement in large, multicenter clinical trials, which
ultimately are required for the validation of potential biomarkers. The costs of such detailed
techniques must also be weighed against the additional information that may be derived
regarding the composition and functional status of the immune infiltrate.
The host immune response to tumors is currently of great interest to oncologists and
researchers following impressive early results of immune checkpoint inhibitor therapy. An
important mechanism of tumor immune evasion is the expression of immune checkpoint
molecules such as CTLA-4 and PD-L1, both on tumor cells and on infiltrating immune cells
[28]. By blocking these signaling pathways, immune checkpoint inhibitors can re-activate
the host immune system to recognize and control the tumor [28]. Clinical trials have
demonstrated often durable responses in different tumor types including melanoma [29,30],
urothelial carcinoma [31], Hodgkin lymphoma [32], non-small cell lung carcinoma [33–35],
renal cell carcinoma [36], and squamous cell carcinoma of the head and neck [37]. However,
responses within tumor types vary widely and the selection of patients likely to respond
remains problematic [38]. Despite FDA approval as companion and complementary
diagnostics for the use of anti-PD-1 therapy in non-small cell lung carcinoma [39,40],
immunohistochemical identification of PD-L1 expression on tumor cells and/or immune
cells is an imperfect biomarker [40] and a significant research effort is ongoing to identify
reliable, broadly applicable and clinically valid biomarkers. T cell infiltration into tumors is
critical to the success of immune checkpoint blockade [41], and tumors with high levels of
infiltrating effector T cells, as measured by gene expression profiling, appear to show
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improved responses [42–44]. Assessment of TILs in this context is a highly active area of
research and guidance for a standardized methodology is therefore timely.
Proposal for a standardized methodology for scoring TILs in solid tumors,
in both primary and metastatic settings
Much research has been performed to establish the prognostic and predictive significance of
TILs in different solid tumors. However, further work is needed to ensure that the valuable
information that could be obtained from TILs assessment is not lost due to issues of resource
commitments, methodology, or lack of standardization. It is our view that a semi-
quantitative H&E based TILs assessment provides clinically relevant information in a format
that is applicable to large-scale randomized clinical trials, to pre-clinical and clinical
research projects and to everyday pathology practice, whether it be in high, middle, or low-
income countries. It needs to be emphasized that the sophisticated tools mentioned above are
resource-intensive and may be difficult to implement in middle or low-income settings. A
biomarker based on a plain H&E stained section is affordable and accessible, and will not
add to the often-restrictive costs of accessing therapy. This is an important consideration in
the current era of immunotherapeutics.
Over the past few years, Roberto Salgado, Sherene Loi and Carsten Denkert have developed
the International Immuno-Oncology Biomarker Working Group on Breast Cancer, with
members including important clinical research groups, pathologists, clinicians and
statisticians worldwide currently knowledgeable in the field of immuno-oncology
biomarkers. The purpose of this Working Group is to develop, in a timely manner, standards
on the assessment of immuno-oncology biomarkers to aid pathologists, clinicians and
researchers in their research and daily practice. The group has, for example, developed the
first International Guidelines on TIL-Assessment in Breast Cancer [14] and other guideline
papers are in development. In addition to the breast cancer experts already included,
academic expert groups from other fields and biomarker expert groups from industry were
contacted and enthusiastically agreed to be a part of this initiative. We include worldwide
representatives of known clinical research groups from expert centers across all continents
(US, Europe, Australia, the Middle East and Japan) and the member list is growing.
For these papers, a panel of pathologists, medical oncologists, biostatisticians and
translational researchers from different expert groups conducted a systematic review of the
literature. Panel members have had experience in TIL assessment in pre-clinical research,
clinical trials or are involved in translational research focused on the interactions between
immunology and cancer. There are no existing guidelines on TIL assessment in solid tumors
available for comparison; neither are there proficiency testing data available from
international organizations. No specific funding was obtained for this project. The
methodology we propose is based on the International Guidelines on TIL Assessment in
Breast Cancer [14], following robust evidence from prospective retrospective phase III trials
in breast cancer. Reviewed studies were not limited to randomized trials, but also included
consecutive and retrospective series and in-press publications. Ways in which this
methodology could be adapted to different tumor types, while remaining as standardized as
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