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From the Division of Experimental Oncology 1,*
Centro di
Riferimento Oncologico, Aviano; the Section of Anatomic
Pathology,
the Department of Experimental and
Diagnostic Medicine, University of Ferrara, Ferrara; the Division of
Clinical Oncology,¶
S. Anna Hospital, Ferrara; the
Department of Pathology,
City Hospital,
Belluno; and the Analytical Epidemiology
Section,
Epidemiology Unit, Centro per lo
Studio e la Prevenzione Oncologica, Firenze, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different genetic pathways have been identified along which colorectal
cancer develops and progresses by a stepwise accumulation of widespread
molecular alterations.5,6
Recent evidence indicates that a
subset of colorectal tumors are characterized by microsatellite
instability (MSI), a genetic defect leading to the progressive
accumulation of insertion/deletion mutations at simple repeated
sequences (microsatellite sequences) as a consequence of impaired
postreplicative DNA mismatch repair.7,8
Patients with
hereditary nonpolyposis colorectal cancer carry germline mutations of
genes involved in DNA mismatch repair and these alterations are
responsible for this mutator phenotype.9
Furthermore, MSI
is also found in 
10 to 20% of sporadic colorectal cancers showing
inactivation of DNA mismatch repair genes by somatic mutations or
epigenetic silencing.10,11
Colorectal tumors with
high-frequency MSI (MSI-H), both hereditary nonpolyposis colorectal
cancer-related and sporadic, are characterized by preferential location
in the proximal colon, diploid nuclear DNA content, high prevalence of
mucinous and medullary histotypes, and poor
differentiation.12,13
Moreover, recent studies reported
that patients with MSI colonic cancers have a better clinical
course.14,15
One possible explanation for the favorable
outcome of these patients is that the enhanced mutation rate associated
with MSI, besides favoring mutations at genes critical for oncogenesis,
may also induce a mutation burden no longer compatible with tumor cell
survival. On the other hand, the peculiar genetic instability of MSI-H
tumors may also lead to a continuous production of abnormal peptides
that, by acting as neoantigens, could elicit specific antitumor immune
responses potentially effective in limiting tumor growth and/or spread.
In support of this hypothesis is the pronounced inflammatory infiltrate
with a high content of tumor infiltrating lymphocytes, observed in
MSI-H colorectal carcinomas.16
Moreover, the presence of
an antitumor cytotoxic immune response may have a favorable impact on
the clinical outcome of patients with colorectal carcinomas. In fact,
irrespective of the presence of MSI, a high content of cytotoxic
effectors infiltrating these tumors strongly correlated with better
survival rates.17,18
Activation of CD8+ cytotoxic lymphocytes (CTLs) is characterized by the expression of peculiar proteins, such as perforin and granzyme B, involved in the induction of apoptosis in target cells. We have recently reported that MSI-H colorectal carcinomas carried significantly higher numbers of activated cytotoxic intraepithelial lymphocytes (IELs) than low frequency MSI (MSI-L) or microsatellite stable (MSS) tumors.19 Moreover, evidence consistent with the presence of local antitumor cytotoxic immune responses in most MSI-H cases was reported.19 On these grounds, it seems of relevance to assess whether the number of activated cytotoxic IELs is related to improved prognosis in patients with MSI-H colonic tumors. The present study was designed to address this issue and, in particular, to verify whether the combined evaluation of MSI and number of activated cytotoxic IELs may allow a more precise prediction of the clinical outcome of patients with stage II and III sporadic colon carcinomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Between January 1988 and December 1992, 245 consecutive patients
with stage II and III colon carcinomas underwent radical surgery at a
single institution (Arcispedale S. Anna, Ferrara, Italy). Among these,
118 showed disease located in the proximal colon, ie, from caecum to
splenic flexure, were included in the study. In nine cases tumor tissue
was not available. Selection for proximal colon cancer cases was
justified by the need to enrich in MSI-H cases, because 90% of
these tumors are located in the right portion of the
colon.14
Adjuvant monochemotherapy with 5-fluorouracil was
administered to 22 patients (7 patients with stage II disease and 15
with stage III disease). Family history of colorectal cancer was
excluded in all cases except one, showing early onset disease and a
first-degree relative with colorectal neoplasia, although the Amsterdam
criteria for hereditary nonpolyposis colorectal cancer were not
fulfilled.20
DNA ploidy analysis was performed in 47 tumor
samples for which frozen tissue was available, as previously
described.21
All patients were followed-up until death or
until December 1998. Information about the clinical outcome was
obtained by hospital chart review or direct telephone interview with
the patients’ personal physicians. Periodic controls included clinical
examination, blood test (including serum carcinoembryonic antigen),
endoscopy, abdominal ultrasonography, and radiography of the thorax.
Computed tomography and magnetic resonance imaging were also performed
when tumor recurrence was suspected.
Analysis of MSI
DNA was extracted from formalin-fixed, paraffin-embedded or frozen tissue, when available. Tumor specimens were chosen by microscopic examination, carefully avoiding areas enriched in nonneoplastic cells and necrotic debris. Tumor and corresponding normal DNA samples were analyzed for MSI at 5 to 10 microsatellite loci. Polymerase chain reaction was performed as previously described.22 In agreement with Boland and colleagues,23 all samples were evaluated for MSI at BAT25, BAT26, D17S250, D2S123, and D5S346, as a first reference panel. A further five microsatellites, including BAT40, D10S197, D18S58, D18S69, and L-myc, were analyzed when only one locus from the first panel showed MSI or when one or more loci were not evaluable. Tumors were scored as having MSI-H when additional bands were present in the polymerase chain reaction products from at least two loci of the first panel or from at least 30% of all examined loci, if additional microsatellites were analyzed. Tumors were scored as having MSS/MSI-L when any (MSS) or <30% (MSI-L) of the examined loci showed additional bands.
Histopathological Analysis
Conventional histopathological parameters, including AJCC/UICC TNM stage, tumor type, and grade of differentiation, were independently assessed by two pathologists (Giovanni Lanza and Roberta Gafá) without any knowledge of the results of genetic analysis. Tumors were typed as adenocarcinomas or mucinous adenocarcinomas according to the criteria of the World Health Organization.24 Poorly differentiated adenocarcinomas with a predominantly solid growth pattern (at least 70% of the tumor area) and lack of marked nuclear pleomorphism were classified as medullary adenocarcinomas.25 Grade of differentiation (well/moderate or poor) was determined following the World Health Organization guidelines.24
Immunohistochemical Analysis
Immunohistochemical analyses were performed as previously described.19 The primary antibodies used were: anti-CD3 (polyclonal, 1:1000) and anti-CD8 (clone cd8/144b, 1:25), purchased from DAKO (Glostrup, Denmark); and anti-granzyme B (clone GrB7, 1:20) obtained from Monosan (Leiden, The Netherlands). The number of IELs positive at immunohistochemistry as well as the number of tumor cells, assessed by morphology, were recorded in the same microscopic fields using a video-assisted cell analysis system (MicroImage; SC Casti Imaging, Venezia, Italy). The ratio between the number of positive IELs and that of tumor cells associated with was then calculated. At least five randomly selected high-power microscopic fields were evaluated, accurately avoiding necrotic and superficial areas. In a limited number of cases, immunohistochemical evaluation was performed on sections from two different blocks.
Statistical Analysis
The index date for survival calculation was defined as the date of diagnostic confirmation for colon cancer. The months of observation were calculated from the index date to the date of last information/death. The observed survival was estimated using the Kaplan and Meier product limit method, and cumulative survival probability was calculated at 5 years stratified according to MSI phenotype and stage at diagnosis. Differences were tested using the log-rank test. To assess the relative excess risk of death/recurrence according to MSI status and type of activated cytotoxic IELs and to control for confounding factors (age as continuous variable, gender, and pathological stage), proportional hazards models were fitted by computing hazard ratios and the corresponding 95% confidence intervals (95% CI). Mean cell count was analyzed dividing the distributions of CD3+, CD8+, and granzyme B+ IELs into two groups of equal size, using the median value as cut-point. The proportional assumption was examined with log-log survival plots or by adding time-dependent interaction terms into the model. Interaction terms between MSI phenotype and type of activated cytotoxic IELs divided according to the median value were calculated by means of the Wald chi-square test with one degree of freedom.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forty-seven patients (43.1%) showed MSI-H tumors, and 62 patients
(56.9%) had MSS/MSI-L tumors (48 MSS and 14 MSI-L). MSI-H tumors were
more frequent in patients older than 60 years (87.2% versus
69.4%; P value, 0.03) than MSS/MSI-L tumors, whereas no
difference in terms of sex and stage distribution was observed (Table 1)
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The median follow-up time among patients alive as of their last follow-up was 74 months (range, 50 to 120 months) and 78 months (range, 46 to 108 months) for patients with MSS/MSI-L and MSI-H tumors, respectively. Adjuvant chemotherapy was administered only to a fraction of patients (22 of 109, 20.2%), the majority of which had stage III disease (15 of 22, 68.1%); cases with MSI-H were not differently treated compared to cases with MSS/MSI-L tumors (19.2% versus 21.0%).
Any difference in the distribution of clinical and pathological features was found between MSS and MSI-L cases.
Phenotypic Characterization of Tumor-Associated IELs
In all cases, IEL density was fairly uniform throughout the
nonnecrotic areas of tumor samples. No correlation was found between
the number of IELs and sex, age, and TNM stage, except for a borderline
association between stage and CD8+ IEL number,
with a slightly lower prevalence among TNM stage III cases (data not
shown). MSI-H tumors carried a higher number of
CD3+ intraepithelial T lymphocytes than MSS/MSI-L
tumors (mean SD: 0.057-0.043 versus 0.043-0.025;
P value, 0.05). Accordingly, a significantly higher
prevalence of CD8+ IELs was observed in MSI-H
than in MSS/MSI-L tumors (mean SD: 0.049-0.045 versus
0.033-0.023; P value, 0.02), indicating a more efficient
recruitment of cytotoxic precursors. Of note, a large proportion of
these cytotoxic precursors was activated, as shown by the higher
content of GrB+ IELs observed in MSI-H cases
(mean-SD: 0.020-0.01 versus 0.012-0.01; P value,
0.0001) (Figure 1)
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Of the 109 patients investigated, 72 (66%) were still alive at the end of the study, with a mean follow-up of 78 months (range, 46 to 120 months). During the observation period, 37 deaths were recorded, 17 of which occurred in the first 2 years from diagnosis. The overall survival probability was 0.85 at 2 years and 0.65 at 5 years of observation. Patients with MSI-H tumors had a better clinical course, with significantly higher 5-year overall survival (82% versus 53%; P value, 0.001) and disease-free rates (82% versus 55%; P value, 0.002) than patients with MSS/MSI-L.
Stratification according to TNM staging further confirmed these
findings. In fact, MSI-H patients with stage III disease had
significantly better overall survival (72%
versus 35%; P value, 0.006) and
relapse-free rates (66% versus 39%; P value,
0.02) than the corresponding MSS/MSI-L patients (Table 2)
. The occurrence of MSI-H was also
correlated with improved clinical outcome in patients with stage II
disease, although differences were not statistically significant, as
shown by 5-year overall survival (89% versus 75%;
P value, 0.2) and relapse-free rates (93% versus
75%; P value, 0.08) (Table 2)
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. Prognostic Value of the Content of Activated Cytotoxic IELs
The prognostic significance of the number of activated cytotoxic
IELs was first investigated by univariate survival analysis,
classifying each case as having high or low CD3+,
CD8+, and GrB+ cell count
according to the median value. Results showed that high levels of
CD3+, CD8+, and
GrB+ IELs were associated with improved survival
(Table 3)
. Multivariate analysis also
confirmed that the content of CD3+,
CD8+, and GrB+ IELs
positively affected the clinical outcome, independently of age, sex,
and TNM stage (Table 3)
.
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. All
interaction terms estimated between MSI phenotype and
CD3+, CD8+, and
GrB+ cells subdivided according to the median
were highly significant.
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90%) compared with the reference category (MSS/MSI-L cases with low
CD3+, CD8+, and
GrB+ cell count). Consistently, patients carrying
tumors with both MSI-H and high GrB+ cell
counts had significantly more favorable 5-year overall survival (94%
versus 51%; P value, 0.0001) (Figure 2A)
and relapse-free rates (94%
versus 53%; P value 0.0001) (not shown) than all
of the other patients. Of note, when only stage III cases were
investigated, differences between the two subgroups were even more
pronounced, with 5-year overall survival (93% versus 34%;
P value, 0.001) (Figure 2B)
and relapse-free rates (86%
versus 36%; P value, 0.001) (not shown)
significantly better in patients with both MSI-H and high content of
GrB+ IELs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most relevant finding comes from the combined evaluation of MSI and
the content of activated cytotoxic IELs. Such an approach allowed the
identification of patients with both MSI-H tumors and high numbers of
activated cytotoxic effectors as a distinct subset of cases
characterized by a very good clinical course. These patients,
comprising 30% of our series of right-sided colonic carcinomas, had
a risk of death of 10% of that observed in patients with MSS tumors
carrying low numbers of activated cytotoxic IELs, independent of TNM
stage, sex, and age. Of note, a quasi-multiplicative interaction in
favorably influencing the clinical outcome was found between MSI-H and
high numbers of activated CTLs infiltrating cancer cell nests.
Consistently, this interaction resulted in markedly improved overall
survival and relapse-free times in the whole series, and these
differences were even more pronounced in patients with stage III
disease.
The finding that only MSI-H cases with a high content of activated cytotoxic IELs have a better prognosis further supports the hypothesis that the presence of local antitumor immune response is probably one of the major determinants of the more favorable course observed in patients with MSI-H tumors. Therefore, it is likely that in most MSI-H cases, the continuous production of abnormal peptides favored by the inherent genetic instability of tumor cells is responsible for the induction of clinically relevant cytotoxic immune responses.
Our findings, suggesting the less aggressive clinical behavior of MSI-H
colonic tumors is likely because of a better local immune control of
the disease, may have relevant implications for the management of these
patients. In fact, our observation that cases showing both MSI-H and a
high content of activated cytotoxic IELs have a very favorable
prognosis questions the need for adjuvant chemotherapy in these
patients. This is a very controversial issue. In fact, recent articles
reported opposite effects of chemotherapy in MSI-H colorectal cancers.
In particular, a recent large retrospective study indicated that
patients with MSI-H Duke’s stage C colorectal cancer did not have a
favorable outcome unless they had received adjuvant
chemotherapy.26
Furthermore, Hemminki and
colleagues27
reported a better prognosis in patients with
MSI-H cancer who had received adjuvant therapy. Nevertheless, in the
first work patients were stratified according to the Duke’s staging
system and therefore it cannot be ruled out that some MSI-H cases with
distant metastases may have not received any additional therapy after
surgery. It is worth considering that, in the present series,
additional therapy was administered only to a minority of cases
(20%), with an even distribution of stage II and III disease in
patients with either MSI-H or MSS/MSI-L tumors. In keeping with our
data, Wright and colleagues28
showed that, in patients
with locally advanced colorectal cancer who have not received
additional chemotherapy, MSI-H is an independent positive prognostic
factor. In addition, recent in vitro evidence indicates that
MSI-H colon cancer cell lines are resistant to 5-fluorouracil at
concentrations similar to those achieved in patients’
tissues.29
On these grounds, our results provide the
rationale for further studies aimed at defining the real usefulness of
adjuvant chemotherapy in the treatment of most patients with MSI-H
colon cancer, particularly in stage III disease.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from Associazione Italiana per la Ricerca sul Cancro.
Accepted for publication March 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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