Review

The adaptive immune response to colorectal cancer: From the laboratory to clinical
practice
N.J. Curtis
a,b,
*
, J.N. Primrose
a,b
, G.J. Thomas
b,c
, A.H. Mirnezami
a,b
, C.H. Ottensmeier
b
a
Department of Surgery, University Hospital Southampton, Tremona Road, Southampton, Hampshire SO16 6YD, UK
b
Cancer Sciences Division, University of Southampton, Somers Cancer Research Building, MP824, Southampton General Hospital,
Southampton SO16 6YD, UK
c
Department of Cellular Pathology, University Hospital Southampton, Tremona Road, Southampton, Hampshire SO16 6YD, UK
Accepted 24 May 2012
Available online - - -
Abstract
Introduction: Analysis of the adaptive immune system in the microenvironment of colorectal cancer is suggested to offer new insights into
tumour biology and prognostic information independent of TNM staging. We aimed to review recent ndings to investigate the potential for
clinical use.
Methods: Relevant papers were identied through online searches regarding tumour inltrating lymphocytes (TIL) in colorectal cancer.
Identied papers were studied, focusing on clinically applicable uses for TIL data in the management of colorectal cancer.
Findings: The majority of identied studies were retrospective and observational in nature. The widest TIL investigation was in post re-
section prognosis but TIL subtypes, counts and methodology showed variability between studies. Recent reports explored TIL in predicting
response to adjuvant and neoadjuvant treatments.
Conclusion: An increasing body of evidence supports that visibility of colorectal cancer to immune attack is substantial and that it limits
disease progression. Analysis of the adaptive immune inltrate in resected colorectal cancer specimens offers prognostic information which
is independent of conventionally measured parameters and potentially superior in predictive value.
2012 Published by Elsevier Ltd.
Keywords: Colorectal cancer; Lymphocytes; Adaptive immune system; Prognosis; Risk stratication
Introduction
The adaptive immune system has long been suspected to
play a key role in the host response to solid tumours. Obser-
vations that chronic inammatory conditions are risk fac-
tors for a variety of cancers
1
and cancer incidence
increases in immunosuppressive states
2
support the concept
that tumour development and host immunity are inextrica-
bly linked. Solid tumours are frequently inltrated by mul-
tiple components of the immune system.
3,4
A high number
of tumour inltrating T lymphocytes (TIL) has been linked
with a favourable outlook in colorectal cancer,
5e8
and the
observations from individual studies were conrmed in a re-
cent meta-analysis.
9
High TIL levels are associated with fa-
vourable prognosis in other solid tumours, identifying the
protective effect of adaptive immunity as a principle, with
relevance for cancer biology in general.
10
Given the initial reports were made twenty ve years
ago,
6,7
it is surprising that evaluation of TIL in primary can-
cers has, to date, had little impact on routine practice. How-
ever, current evidence linking the presence of TIL with
outlook in colorectal cancer, combined with modern tissue
processing and laboratory techniques sees us poised to ex-
ploit this information for the benet of patients. This review
summarises recent ndings and highlights translational ap-
plications that can inuence clinical decision making in co-
lorectal cancer.
* Corresponding author. Cancer Sciences Division, University of South-
ampton, Somers Cancer Research Building, MP824, Southampton General
Hospital, Southampton SO16 6YD, UK. Tel.: 44 2380 777222x8632.
E-mail address: nathancurtis@doctors.org.uk (N.J. Curtis).
0748-7983/$ - see front matter 2012 Published by Elsevier Ltd.
doi:10.1016/j.ejso.2012.05.011
Available online at www.sciencedirect.com
EJSO xx (2012) 1e8 www.ejso.com
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
Methods
Studies relating to TIL were identied by performing
a Pubmed search using the keywords: Colorectal cancer, tu-
mour inltrating lymphocytes, adaptive immune system,
prognosis and risk stratication. The search was restricted
to English language publications since 1980. The reference
lists of relevant papers were examined for further studies.
Focus was given to studies reporting TIL with clinical
and oncological outcomes.
The adaptive immune system and its response to
cancer
T lymphocytes differentiate into a variety of mature sub-
types with differing and complementary functions. TIL can
be characterised by the analysis of their cell surface mole-
cules, usually given standardized names using CD (cluster
determinant) followed by a number. CD3 is present on all T
lymphocytes; CD8 marks effector T cells whereas CD4 is
displayed on helper T lymphocytes. Galon and colleagues
showed that a high number of CD3

cells in colorectal tu-

mour tissue was associated with a markedly improved prog-
nosis,
5
compared to patients in whom these TIL were rare,
a decreased relapse rate, longer disease free and overall sur-
vival (Fig. 1). The CD3 count was independent of TNM
staging, and within all stages patients with low number of
TIL showed signicantly worse clinical outcomes
5
(Fig. 1).
All nucleated cells turn over proteins by synthesis and
degradation into small peptide fragments. In the cytoplasm
these peptides may then be associated with major histocom-
patability complex (MHC) I and II and transported to the
cell surface for display to the extracellular environment.
Circulating T lymphocytes sample these peptides and the
outcome of this interaction determines the cellular fate: if
the T cell recognizes the peptide in the MHC I groove
through its unique T cell receptor (TCR) it becomes acti-
vated and ultimately eliminates the target cell e the T
cell has taken on cytotoxic or killer function. T cells re-
circulate and expand more quickly at the next encounter
of antigen. In contrast, if the TCR does not bind to any
MHC/peptide complexes the T cell remains quiescent.
The overall result is maintaining host integrity by identify-
ing abnormal cells, for example following viral infection
3
or to control cells that have been altered through carcino-
genic processes
4
(Fig. 2). Higher CD8

cytotoxic TIL
counts are linked to similar prognostic benets as raised
TIL CD3

counts
5
(Fig. 3).
The TCR-antigen interaction triggers T cell division
with some T cells becoming memory cells. The presence
of the surface molecule CD45RO (CD45RO

) indicates
a memory T cell that has been activated following success-
ful exposure to antigen.
11
High CD45RO

counts in the tu-

mour are associated with an improved prognosis,
12
arguing
that these cells are functionally active and not merely by-
standers. In keeping with this concept, a recent series shows
that compared to matched, normal bowel mucosa, colorec-
tal cancers contain a higher proportion of activated and cy-
totoxic CD8

cells
11
and that these T cells can recognize
antigen from colorectal cancer cells. CD45RO

cell counts
remained an independent prognostic marker when different
molecular genotypes characterized by KRAS or BRAF mu-
tations and DNA microsatellite instability (MSI) were con-
sidered.
13
The presence of MSI is a hallmark of the loss of
DNA mismatch repair function and interestingly, MSI is as-
sociated with increased TIL and confers a favourable prog-
nosis.
14
It is hypothesised therefore that loss of mismatch
repair results in the development of new non-self, tumour
associated antigens which provoke an adaptive T-cell re-
sponse.
14
Experimental evidence for this concept has re-
cently been brought in a mouse tumour system.
15
Molecular examples of human epiptopes resulting from
frameshift mutations have been reported
16,17
and shown
to be immunogenic to CD8

cytotoxic T lymphocytes.
18
A particular mutation has been detected in 82% of MSI
cancers in the gene U79260(FT0).
19
This mutation gener-
ates a new peptide TLSPGWSAV, which can be visible to
CD8 T cells in patients with the common MHC I allele
HLA A020*. Such data not only provide proof of principle
to link genetic events to immunological outcomes but also
offer a new target that could be exploited by vaccination.
19
Further evidence for the role of the adaptive immune
system was obtained through the study of the subtypes of
helper (CD4

) T lymphocyte population, which control

the subsequent immune reaction through the secretion of
cytokines. T helper class I (Th1) are strong stimulators of
cell mediated immunity
20
and cytotoxic T lymphocytes
through the production of cytokines, such as interferon
gamma.
21
High levels of mRNA for Th1 associated genes
were strongly associated with a low relapse rate and longer
disease free survival than low mRNA levels.
5,20
In contrast,
the Th2 phenotype favours recruitment of the innate im-
mune system and humoural immunity with suppression of
antigen presenting cells and, in terms of antitumour immu-
nity, appears to be less effective than a Th1 response. A
fourth T cell subtype, CD4

CD25

regulatory T cells,
Figure 1. Marked differences in disease free survival are seen with each
stage dependent on CD3 counts alone. A low T cell inltrate is a poor
prognostic factor across all stages including early disease.
**p < 0.0001. Used with permission.
5
2 N.J. Curtis et al. / EJSO xx (2012) 1e8
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
suppress the activation of Th1 type cytotoxic T lympho-
cytes and are characterised by expression of the nuclear
transcription factor, FoxP3.
22
These cells are linked with
worse outcomes in ovarian,
23
pancreatic
24
and hepatocellu-
lar
25
carcinoma but, in contrast have been reported as a pos-
itive prognosis indicator in colorectal cancer.
12,26
This
highlights that our understanding of the complex interplay
between tumour and host remains incomplete.
It has been suggested that both the immune inltrate and
tumour stage have an inverse relationship and evolve over
time in a linked, parallel fashion. In order for the cancer
to progress, tumour cells need to evade the host immune re-
sponse.
27
Schreiber and Old described how immune attack
will initially eliminate cancer cells but this selective pres-
sure will promote survival of tumour cells with reduced im-
munogenicity. After a period of equilibrium,
28
further
immunoediting could be expected to facilitate eventual tu-
mour immune escape with adverse results for the patient.
29
Consistent with this, more advanced tumour stages have
a lower overall TIL count with lower numbers of CD3
13
and CD8
30
cells (Fig. 4). This correlated with reduced dis-
ease free survival and higher relapse rates.
31
However, per-
haps the most important nding to date is that a lower TIL
inltrate, irrespective of TNM stage, is strongly associated
with colorectal cancer recurrence. This is independent of
standard histopathology staging data.
5
Analysis of various
cancers has reproduced this nding suggesting evaluation
of TIL will prove useful across a wide range solid
tumours.
32
Presence of high CD8

and CD45RO

TIL
counts are additionally associated with the absence of ex-
tramural venous, lymphatic and perineural invasion.
33
This absence of the histopathological signs of early meta-
static invasion was linked with a marked long term survival
benet in colorectal malignancy.
33
Immune scoring
The creation of clinically applicable scoring methods
has been attempted to bring the immunological ndings
into routine use. The initial report on TIL in rectal cancer
from Jass
7
came as part of an attempt to create a prognostic
classication. Klintrup and Shia have also proposed scores
but these too have not reached routine use.
34,35
Building on
their groups previous observations, Pages
8
proposed a sim-
ple score based on the immune inltrate in each tumour. In
stage I and II colorectal cancers, at both the centre of the
tumour and the invasive margin, both CD8

and CD45RO

TIL were quantied. A score of one was given for each of

these four tests if the number of cells present was above the
median and zero if below. A uniformly high inltrate would
score the maximum of four and a universally low inltrate
would score zero. A striking correlation with ve year out-
comes was seen
8,33
(Fig. 5). Within tumour stages the im-
mune score stratied an individuals risk of recurrence.
This would not have been evident from pTNM staging
alone and routine application is attractive as it offers
Figure 2. Simplied cellular mechanisms of the adaptive T cell response to colorectal cancer. 1 e DNA alterations in the cancer cell result in abnormal
antigens derived from altered proteins being displayed on the cell surface. 2 e T lymphocytes sample these peptides. Recognition of non-self, abnormal
antigen leads to activation. 3 e The activated cell undergoes clonal expansion through interaction with T helper lymphocytes. 4 e Recognition of the ab-
normal antigen leads to lysis of the cell by cytotoxic CD 8 T lymphocytes.
3 N.J. Curtis et al. / EJSO xx (2012) 1e8
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
a simple stratication tool to reduce the wide variety of out-
comes that exists within disease stages.
Practicalities of immune inltrate testing
Analysis can be performed using standard immunohisto-
chemistry techniques without any special equipment. TIL
within each sample are quantied manually or utilising au-
tomated image analysis software meaning results can be
available at the same time as conventional histopathologi-
cal evaluation, to help inform multi-disciplinary discus-
sions. In the research setting, large numbers of samples
can rapidly be processed on tissue microarrays
36
but this
tool is difcult to establish and validate for routine clinical
use. Combined analysis of the tumour centre and invasion
margin has been suggested to overcome sampling bias
from heterogeneity in the tissue distribution of T cells,
but debate exists about which part of the tumour and num-
ber of samples is optimal. The testing of multiple sites
within tumours (and duplication of testing) seems
pragmatic as colorectal cancers can be architecturally, mo-
lecularly and biologically heterogeneous.
TIL quantication has also been performed using quar-
tiles
13
and continuous scales
37
rather than utilising the me-
dian value but these tools have not found wide acceptance.
More complex analyses examining ratios of effector/helper
T cells (CD8/CD4) or effector/regulatory cells (CD8/
FoxP3) have been performed
9
although it remains unclear
whether these ratios are more effective prognosticators
than CD8

and CD45RO

cell counts. Pragmatically and

for clinical use a simple scoring system would be most at-
tractive and well within the skill set available in any histo-
pathology laboratory.
Potential uses of immune information in colorectal
cancer management
Surgical resection of colorectal cancer still offers the best
chance of achieving cure but immune scoring has real poten-
tial to impact on post operative management decisions as an
Figure 3. Top left e Patients that did not relapse (white bars) had higher cell counts than those that developed metastatic disease (black bars). Top right e
Higher counts were strongly associated with a longer overall survival. Bottom e Disease free survival did not alter by site of the CD3 TIL but when com-
bined the prognostic strength was improved (n 415) **p < 0.0001 Adapted with permission.
5
4 N.J. Curtis et al. / EJSO xx (2012) 1e8
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
adjunct to established staging. Currently around 25% of
stage II patients are expected to relapse
8
but would not rou-
tinely be offered adjuvant chemotherapy in the UK. An im-
mune score derived risk stratication model could be utilised
to identify those patients who should receive adjuvant treat-
ment even in the group traditionally considered to have
a good prognosis groups.
31
It should be noted that adjuvant
chemotherapy outcomes have not been correlated with TIL
inltrates to date. TIL densities in early disease may become
more important as the introduction of bowel cancer screen-
ing should result in a trend towards the earlier detection and
treatment of colorectal malignancy.
The addition of immune score information is also likely
to be useful for deciding follow up strategies, with resulting
benet in health economic terms. A patient at low risk
based on conventional staging, with clear resection margins
and favourable TIL prole could be offered a less intensive
follow up protocol without additional risk or may even not
require follow up at all. Clearly randomized study of this
question needs to precede clinical implementation.
Resection of locally recurrent and distant colorectal can-
cer metastases is increasing in the UK. Assessing TIL in the
resected primary cancer as a tool for predicting risk of local
relapse will build the necessary datasets to inform the deci-
sion making when contemplating further resection. In addi-
tion to current understanding of the molecular biological
changes than allow distant tumour spread, metastatic dis-
ease also represents the failure of immune control of the tu-
mour. The presence, role and prognostic use of TIL within
metastatic tissue has not been thoroughly investigated. This
area requires urgent study as it now appears possible that
resection of metastatic disease in the presence of a low im-
mune inltrate within the primary colorectal tumour is un-
likely to be a curative procedure.
38
To date, the analysis of immune parameters has not been
exploited to stratify patients for immunotherapy. Inducing
an adaptive immune response by vaccination or using im-
munostimulatory antibodies is an attractive strategy and
in melanoma, prostate cancer and lung cancer are within
reach of standard clinical practice.
4
Vaccine trials in colo-
rectal cancer also demonstrate that immune responses can
be induced by vaccination
39
but whether this translates
into clinical benet is the subject of intense study, both in
the UK and worldwide. Thus far, immunotherapy trials
have focused on patients with advanced disease. While
this approach allows rapid efcacy evaluation, the decrease
in TIL seen in advanced cancer represent an inherit draw-
back; additionally the tumour can be expected to have suc-
cessfully evaded the host response.
11
Therefore the highest
vaccine immunogenicity and clinical impact is to be ex-
pected in patients with completely resected disease.
Future potential applications of TIL information
Rather than only using TILdata to identify patients at high
risk of relapse, an intriguing possibility is that TIL density
both correlates with susceptibility and expected response to
chemotherapy.
40
Apoptotic cell death induced by Oxaliplatin
Figure 5. KaplaneMeier graph showing a correlation between immune
score and disease free survival in 602 stages I and II colorectal cancer pa-
tients. A lower immune response is seen to be a negative prognostic factor.
Used with permission.
8
Figure 4. A e A higher T stage is seen to be associated with a lower num-
ber of CD8 cells. In stage II and III disease, the quantication of CD8
cells reveals a signicantly lower inltrate present in cases that go on to
relapse. This can differentiate between low and high risk patients within
each disease stage. Adapted with permission.
30
5 N.J. Curtis et al. / EJSO xx (2012) 1e8
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
has been shown to be immunogenic with enhanced T lym-
phocyte response.
41
Cellular alterations following external
beam radiation or chemotherapy render the cancer more sus-
ceptible to T lymphocyte mediated lysis.
42,43
Predicting re-
sponse is attractive as it may protect a subgroup of patients
from treatment side effects if they are unlikely to benet.
An associated saving in resources may also result. Again for-
mal study of this question is urgently needed at a time when
personalised treatment has become a key agenda point for na-
tional healthcare provision.
TIL data from resection specimens may translate to spe-
cic anatomical sub locations and small biopsy samples.
Yasuda reported a striking correlation in the TIL within
the initial diagnostic biopsies from rectal adenocarci-
nomas.
44
After neoadjuvant therapy and surgical resection,
the histopathological tumour regression grade
45
strongly
correlated with the density of the inltrate and was found
to be an independent prognostic factor for a complete path-
ological response.
44
This novel nding warrants further in-
vestigation and argues that detailed multisite TIL sampling
may not be required for routine application.
There has been little investigation of immune inltrates
seen in metastatic disease. Distant spread has been strongly
associated with low immune inltrates within the primary
tumour; but initial reports have shown wide variations in
the TIL density in resected liver metastases.
46
As in pri-
mary tumours, high TIL count were associated with re-
sponse to chemotherapy and longer time to progression.
46
A high CD8

count was associated with long term survival

following liver resection but interestingly, in contrast to the
primary tumour microenvironment, a high CD4

count was
associated with poor survival.
38
The wide range of out-
comes in stage IV colorectal cancer has led to calls for a re-
classication of advanced disease.
47
TIL data may assist
this process.
46
Immune analysis of metastatic tissue alone
or in combination with the original primary may reveal fur-
ther useful prognostic data. This research is currently being
undertaken within our group.
Future steps and limitations
To date, TIL studies have been retrospective. There is
a clear need for the collection of prospective, multi-
centre, high quality trial data. Initial observational studies
should allow progression to randomised trials where current
practice is compared with the addition of TIL data to man-
agement decisions. This would signicantly strengthen the
applicability of current ndings for routine practice as well
as dening and then validating a standardised, reproducible
TIL scoring scheme. Large patient numbers would further
allow benchmarking of methods for quantifying each cellu-
lar component. If, as predicted, immune scoring is con-
rmed to correlate with clinical outcomes in prospective
studies, the immune score following resection will inu-
ence stratication of patients for adjuvant treatment
decisions and move towards individualisation of patient
management.
As with all biomarker studies, issues around differing
tissue xation, assays, stain and slide heterogeneity have
not been settled for TIL. We note that the ndings of
Gooden and colleagues
9
meta-analysis were made from
a heterogeneous group of studies on multiple tumour types
from a wide geographical area. This provides weak evi-
dence that TIL biology is universal across tumour types
but the reliability of epitope preservation and overall study
reproducibility has not conrmed.
Further clarication is required when specically con-
sidering rectal cancers. Whilst it is clear that rectal cancer
has TIL
7
few papers report tumour locations or whether
outcomes differed between colonic and rectal adenocarci-
nomas within their study cohort. Subgroup analysis would
be informative and address differences arising from ana-
tomical factors and current use of treatment modalities, par-
ticularly neoadjuvant therapy. It is noted that one group
were not able to reproduce their colonic TIL ndings within
their rectal cancer subgroup.
48
We found no reports on the TIL density observed in tu-
mours with acute clinical presentations and resections in
the presence of factors that may be major confounders of
TIL counts. Whilst it could be hypothesised that the overall
cell counts would be high, neutrophil and TIL quantica-
tion is warranted and correlation with outcomes should be
investigated noting that inammation may be detrimental
to oncological outcomes as seen following anastomotic
leak.
49
A consensus that current understanding and experiences
with TIL can inuence clinical practice is emerging. The
Societies for Immunotherapy of Cancer have initiated
a taskforce on immunoscoring and announced interna-
tional workshops to take place this year
50
with the aim
of providing recommendations on assay harmonisation, es-
tablishing collaborations and validating TIL studies. These
practical steps highlight the translational drive behind TIL
data and the push towards its implementation into routine
practice.
Conclusion
An increasing body of evidence supports that visibility
of colorectal cancer to immune attack is substantial and
that it limits disease progression and metastatic potential.
Information derived from analysis of the adaptive immune
inltrate in resected human colorectal cancer specimens of-
fers prognostic information which appears independent of
conventionally measured parameters and potentially supe-
rior in predictive value. We predict that all aspects of man-
agement will be inuenced by this rapidly evolving eld.
Funding
None.
6 N.J. Curtis et al. / EJSO xx (2012) 1e8
Please cite this article in press as: Curtis NJ, et al., The adaptive immune response to colorectal cancer: From the laboratory to clinical practice, Eur J Surg
Oncol (2012), doi:10.1016/j.ejso.2012.05.011
Conict of interest
None.
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