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. 2001 Mar;48(3):360–366. doi: 10.1136/gut.48.3.360

Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status

J Michael-Robinson 1, A Biemer-Huttmann 1, D Purdie 1, M Walsh 1, L Simms 1, K Biden 1, J Young 1, B Leggett 1, J Jass 1, G Radford-Smith 1
PMCID: PMC1760146  PMID: 11171826

Abstract

BACKGROUND—The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response.
AIMS—To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status.
METHODS—The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody.
RESULTS—Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3+ TILs (>75%) also stained with anti-CD8. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p<0.0001). MSI-H cancers showed a twofold higher rate of apoptosis (mean (SD) 3.52 (0.34)%) than the MSS cancers (1.53 (0.23)%) while the MSI-L subgroup had an intermediate level (2.52 (0.35)%) (p<0.0001). Overall, there was a small (r=0.347) but significant linear correlation between CD3+ and M30+ random apoptosis counts (p<0.001). However, TILs and apoptosis showed little colocalisation.
CONCLUSIONS—While TILs might be expected to explain the increased apoptotic rate and improved prognosis of MSI-H cancers, it is likely that TILs and apoptosis are independent characteristics of MSI-H cancers.


Keywords: colorectal cancer; DNA microsatellite instability; tumour infiltrating lymphocytes; apoptosis

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Figure 1  .

Figure 1  

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Distribution of CD3+ tumour infiltrating lymphocytes (TILs) in colorectal cancer. All 102 tumour CD3+ counts are represented by a triangle. The score of 50 CD3+ lymphocytes was chosen as the cut off for TIL positive cancers (broken line) and the horizontal bars represent the mean count for each group. MSS, microsatellite stable; MSI-L, microsatellite instability-low; MSI-H, microsatellite instability-high.

Figure 2  .

Figure 2  

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Example of a typical undifferentiated microsatellite instability-high (MSI-H) cancer heavily infiltrated with lymphocytes. (A) Standard haematoxylin and eosin stained section. (B) Serial section of the same tumour stained with anti-CD3, revealing T lymphocytes. (C) Serial section stained with anti-CD8. Note that most CD3+ T cells also show positive staining with anti-CD8. (D) Serial section of the same tumour stained with anti-CD20, a B cell marker. Note that no intraepithelial cells have stained positively in this section (original magnification ×60).

Figure 3  .

Figure 3  

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Serial sections of a microsatellite instability-low (MSI-L) cancer (A) stained routinely with haematoxylin and eosin and (B) staining with the M30 cytoDEATH antibody specific for epithelial cell apoptosis. The brown cytoplasmic staining indicates the many apoptotic tumour cells present in this field (original magnification ×120).

Figure 4  .

Figure 4  

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Serial sections of a well differentiated colorectal cancer stained with (A) anti-CD3 and (B) M30 CytoDEATH antibody. Note that only one apoptotic cell is present in this area densely infiltrated with tumour infiltrating lymphocytes (original magnification ×120).

Figure 5  .

Figure 5  

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Example of M30 CytoDEATH and CD3 double staining, counterstained with methyl green. The brown DAB staining indicates M30+ apoptotic tumour cells (arrows) and purple Vector VIP staining reveals the CD3+ T cells present in this tumour infiltrating lymphocyte (TIL) positive microsatellite instability-high (MSI-H) cancer. Note that M30+ neoplastic cells and CD3+ TILs do not coincide (original magnification ×120).

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