Reliability of tumor-infiltrating lymphocyte and tertiary lymphoid structure assessment in human breast cancer

Abstract

The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R=0.74) but this did not extend to tertiary lymphoid structures (R=0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies.

Heterogeneity of tumour‐infiltrating lymphocytes in breast cancer and its prognostic significance

“…Additionally, our data showed that CD3+, CD8+, FOXP3+, CD20+ and CD68+ cells were positively correlated with AV‐TILs percentage, and that TILs were higher in the more aggressive TNBC subtype than in other subtypes. It has been reported that TILs assessed in H&E‐stained sections are significantly correlated with CD20 and CD45 positivity in BC . The high‐CD8+ and high‐CD3+ cellular infiltrate showed good prognostic and independent predictive value in TNBC regardless of other clinicopathological parameters.…”

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research

“…The importance of tertiary lymphoid structures, which represent an organized immune infiltrate with all the features of a lymph node to facilitate antigen presentation, lymphocyte education and effector cell proliferation, has been described in different tumor types [13]. A recent study suggests that IHC is required to reliably identify tertiary lymphoid structures [84], however consensus on the most appropriate marker or combination of markers for this task is not yet apparent. Whether further descriptive analysis of the immune infiltrate in tumors, incorporating features such as the density and distribution of TILs, organization into tertiary lymphoid structures, or the presence of hotspots, adds prognostic information to an overall TILs assessment remains to be determined.…”

“…ICC improved to 0.89 (95% CI 0.85–0.92) and it was suggested that visual reference tools such as the software used in this study might be of value both in research and in clinical practice [14,52]. A recent study has shown similarly high inter-observer and intra-observer ICC for scoring TILs in invasive breast carcinoma, however it was noted that agreement for the iTILs scores was lower than for the sTILs scores [84]. There were no significant differences between the inter- and intra-observer ICC values when scoring H&E sections or IHC stained sections, and concordance between H&E and IHC scores was high (ICC 0.75 for iTIL and ICC 0.84 for sTIL) [84].…”

“…The intensity and distribution of TILs in breast cancers is often heterogeneous [82,83] and it is recommended to give an overall assessment rather than focusing on hotspots of intense infiltration, based on a lack of data demonstrating any clinical significance of this heterogeneity [14]. Recently, Mani et al demonstrated more heterogeneity within a single biopsy than between biopsies or regions from a given breast cancer, suggesting that the average lymphocyte score from a single biopsy may be representative of the whole tumor [82], supported by another recent study showing high correlation between TILs assessed on a pre-operative core biopsy and those assessed on the resection specimen [84]. Interestingly, despite the lack of functional information gained from assessing lymphocytes on H&E only, studies using this method have shown similar prognostic results to those separating lymphocyte subsets by immunohistochemistry [14].…”

“…A recent study has shown similarly high inter-observer and intra-observer ICC for scoring TILs in invasive breast carcinoma, however it was noted that agreement for the iTILs scores was lower than for the sTILs scores [84]. There were no significant differences between the inter- and intra-observer ICC values when scoring H&E sections or IHC stained sections, and concordance between H&E and IHC scores was high (ICC 0.75 for iTIL and ICC 0.84 for sTIL) [84]. Further reproducibility studies such as these are important to thoroughly validate TILs as a biomarker, compare different methodologies, and to work towards a consensus on the degree of reproducibility required for TIL assessment and for biomarkers more generally.…”