Verification of the methodology for evaluating tumor-infiltrating lymphocytes in colorectal cancer

The density of tumor-infiltrating lymphocytes (TILs) have been reported to reflect antitumor immune response and correlate with prognosis in malignancy. However, the methodology for evaluating the density of TILs by an immunohistochemical analysis differs among reports. The aim of this study was to verify the methodology for evaluating the density of TILs by immunohistochemical analysis and thereby identify the optimum methodology in clinical setting.

Three-hundred-thirteen patients who underwent curative operation for stage II/III colorectal cancer were enrolled. We retrospectively examined the density of TILs using immunohistochemical staining according to each method as follows: 1) subset of lymphocytes (i.e. CD4⁺/CD8⁺), 2) selected fields (i.e. at random or focusing on hot spots), 3) location in low-power field (i.e. the invasive margin [TILs^IM] or the center of the tumor [TILs^CT] or the surface of the tumor [TILs^ST]), and 4) location in high-power field (i.e. in tumor stroma [sTILs] or intra-tumor cells [iTILs] or total TILs [tTILs: sTILs+iTILs]). We then assessed the prognostic value of the density of TILs^IM evaluated as described above. We also evaluated the correlation between the density of TILs^IM and that of TILs^CT/TILs^ST.

Only the densities of CD8⁺sTILs^IM and CD8⁺tTILs^IM evaluated in randomly selected fields were significantly associated with the survival. Furthermore, the density of CD8⁺TILs^IM was significantly associated with that of CD8⁺TILs^CT and CD8⁺TILs^ST.

We concluded that best and easiest way to evaluate the density of TILs in the clinical setting may be to assess the density of CD8⁺tTILs^IM in randomly selected fields.