The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC. A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches. The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12–18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-β by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-β production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8⁺ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

Purpose: This study tested the hypothesis that the type of dose fractionation regimen determines the ability of radiotherapy to synergize with anti–CTLA-4 antibody. Experimental Design: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice at two separate sites, defined as a “primary” site that was irradiated and a “secondary” site outside the radiotherapy field. When both tumors were palpable, mice were randomly assigned to eight groups receiving no radiotherapy or three distinct regimens of radiotherapy (20 Gy × 1, 8 Gy × 3, or 6 Gy × 5 fractions in consecutive days) in combination or not with 9H10 monoclonal antibody against CTLA-4. Mice were followed for tumor growth/regression. Similar experiments were conducted in the MCA38 mouse colon carcinoma model. Results: In either of the two models tested, treatment with 9H10 alone had no detectable effect. Each of the radiotherapy regimens caused comparable growth delay of the primary tumors but had no effect on the secondary tumors outside the radiation field. Conversely, the combination of 9H10 and either fractionated radiotherapy regimens achieved enhanced tumor response at the primary site (P < 0.0001). Moreover, an abscopal effect, defined as a significant growth inhibition of the tumor outside the field, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy (P < 0.01). The frequency of CD8+ T cells showing tumor-specific IFN-γ production was proportional to the inhibition of the secondary tumor. Conclusions: Fractionated but not single-dose radiotherapy induces an abscopal effect when in combination with anti–CTLA-4 antibody in two preclinical carcinoma models. (Clin Cancer Res 2009;15(17):5379–88)

Purpose: Recent studies suggest that tumor-infiltrating lymphocytes (TILs) are associated with disease-free (DFS) and overall survival (OS) in operable triple-negative breast cancer (TNBC). We seek to validate the prognostic impact of TILs in primary TNBCs in two adjuvant phase III trials conducted by the Eastern Cooperative Oncology Group (ECOG). Patients and Methods: Full-face hematoxylin and eosin–stained sections of 506 tumors from ECOG trials E2197 and E1199 were evaluated for density of TILs in intraepithelial (iTILs) and stromal compartments (sTILs). Patient cases of TNBC from E2197 and E1199 were randomly selected based on availability of sections. For the primary end point of DFS, association with TIL scores was determined by fitting proportional hazards models stratified on study. Secondary end points were OS and distant recurrence–free interval (DRFI). Reporting recommendations for tumor marker prognostic studies criteria were followed, and all analyses were prespecified. Results: The majority of 481 evaluable cancers had TILs (sTILs, 80%; iTILs, 15%). With a median follow-up of 10.6 years, higher sTIL scores were associated with better prognosis; for every 10% increase in sTILs, a 14% reduction of risk of recurrence or death (P = .02), 18% reduction of risk of distant recurrence (P = .04), and 19% reduction of risk of death (P = .01) were observed. Multivariable analysis confirmed sTILs to be an independent prognostic marker of DFS, DRFI, and OS. Conclusion: In two national randomized clinical trials using contemporary adjuvant chemotherapy, we confirm that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. Studies assessing outcomes and therapeutic efficacies should consider stratification for this parameter.