Table 1 Methods and parameters of tumor-infiltrating lymphocytes (TILs) assessment in DCIS

Methods for evaluation of TILs in DCIS

 (1) TILs were assessed in hematoxylin and eosin-stained sections. Only full-face sections from surgically excised specimen were assessed. Lesions diagnosed on core biopsy were not included 

(2) One representative section (4 µm), per patient, which has the largest tumor burden, was selected for TILs assessment 

(3) All mononuclear inflammatory cells apart from polymorphonuclear leukocytes were counted

(4) TILs within the boundaries of the DCIS were assessed. TILs beyond the tumor limits, surrounding normal ducts/lobules, adjacent fatty tissue, lobular carcinoma in situ, regressive hyalinosis, crushed artefacts or sites of previous biopsy were excluded 

(5) TILs within tumor cells (intra-tumor TILs) were not assessed 

(6) TILs were assessed around all malignant ducts up to 20 ducts. For lesions with more than 20 malignant ducts, we assessed TILs surrounding 20 ducts (five ducts from each quadrant of the lesion) 

(7) TILs were assessed around average sized ducts only (case specific). TILs around very large DCIS ducts such as mass forming papillary carcinoma, branching or confluent DCIS ducts or very small ducts such as terminal duct-lobular system involved by DCIS were excluded 

(8) Any TILs infiltrating the ducts’ circumference were considered. Overlapping TILs between adjacent ducts were counted once

Parameters used for TILs assessment^a

 (A) Estimation of stromal TILs (as previously published):

  ● The stromal area was defined as the area surrounding the DCIS duct within two high-power microscopic fields and used for evaluation of stromal TILs percentage [15, 17, 18]. In cases with numerous involved ducts, an evaluation of the area surrounding the whole lesion was performed, and percentage of stromal TILs in the total stromal area of all DCIS involved ducts was determined [5, 15, 19] 

(B) Estimation of periductal TILs (based on counting TILs around all DCIS duct profiles up to 20 ducts)     

(1) Evaluation of the mean number of TILs touching DCIS involved ducts (defined as TILs touching or within one lymphocyte cell thickness from ducts’ basement membrane)  

(2) Evaluation of the mean number of TILs within 0.2 mm distance from the ducts  

(3) Evaluation of mean number of TILs within 0.5 mm distance from the ducts

(4) Evaluation of mean number of TILs with 1.0 mm distance from the ducts  

(5) Evaluation of the TILs hotspot defined by largest number of lymphoid aggregates directly surrounding or located between DCIS ducts within the boundaries of the lesion  

(6) Evaluation of lymphoid follicles formations with reactive germinal centers in the stroma directly surrounding or located between DCIS ducts within the boundaries of the lesion