Purpose: The aim of the current study was to conduct a pooled analysis of studies that have investigated the prognostic value of tumor-infiltrating lymphocytes (TILs) in early-stage triple negative breast cancer (TNBC). Methods: Participating studies had evaluated the percentage infiltration of stromally located TILs (sTILs) that were quantified in the same manner in patient diagnostic samples of early-stage TNBC treated with anthracycline-based chemotherapy with or without taxanes. Cox proportional hazards regression models stratified by trial were used for invasive disease-free survival (iDFS; primary end point), distant disease-free survival (D-DFS), and overall survival (OS), fitting sTILs as a continuous variable adjusted for clinicopathologic factors. Results: We collected individual data from 2,148 patients from nine studies. Average age was 50 years (range, 22 to 85 years), and 33% of patients were node negative. The average value of sTILs was 23% (standard deviation, 20%), and 77% of patients had 1% or more sTILs. sTILs were significantly lower with older age ( P = .001), larger tumor size ( P = .01), more nodal involvement ( P = .02), and lower histologic grade ( P = .001). A total of 736 iDFS and 548 D-DFS events and 533 deaths were observed. In the multivariable model, sTILs added significant independent prognostic information for all end points (likelihood ratio χ², 48.9 iDFS; P < .001; χ², 55.8 D-DFS; P < .001; χ², 48.5 OS; P < .001). Each 10% increment in sTILs corresponded to an iDFS hazard ratio of 0.87 (95% CI, 0.83 to 0.91) for iDFS, 0.83 (95% CI, 0.79 to 0.88) for D-DFS, and 0.84 (95% CI, 0.79 to 0.89) for OS. In node-negative patients with sTILs ≥ 30%, 3-year iDFS was 92% (95% CI, 89% to 98%), D-DFS was 97% (95% CI, 95% to 99%), and OS was 99% (95% CI, 97% to 100%). Conclusion: This pooled data analysis confirms the strong prognostic role of sTILs in early-stage TNBC and excellent survival of patients with high sTILs after adjuvant chemotherapy and supports the integration of sTILs in a clinicopathologic prognostic model for patients with TNBC. This model can be found at www.tilsinbreastcancer.org .

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. Significance: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy. This article is highlighted in the In This Issue feature, p. 1

For primary triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NAC), higher pretreatment tumor-infiltrating lymphocytes (TILs) correlates with increased pathologic complete response (pCR) rates, and improved survival. We evaluated the added prognostic value of residual disease (RD) TILs to residual cancer burden (RCB) in predicting survival post-NAC. We combined four TNBC NAC patient cohorts who did not achieve pCR. RD TILs were investigated for associations with recurrence-free survival (RFS), and overall survival (OS) using Cox models with stromal TILs as a continuous variable (per 10% increment). The likelihood ratio test was used to evaluate added prognostic value of RD TILs. A total of 375 RD TNBC samples were evaluable for TILs and RCB. The median age was 50 years, with 62% receiving anthracycline/taxane chemotherapy. The RCB class after NAC was 11%, 50%, and 39% for I, II, and III, respectively. The median RD TIL level was 20% (IQR 10-40). There was a positive correlation between RD TIL levels and CD8+ T-cell density (ρ = 0.41). TIL levels were significantly lower with increasing post-NAC tumor (P = 0.005), nodal stage (P = 0.032), but did not differ by RCB class (P = 0.84). Higher RD TILs were significantly associated with improved RFS (HR: 0.86; 95% CI 0.79-0.92; P < 0.001), and improved OS (HR: 0.87; 95% CI 0.80-0.94; P < 0.001), and remained significant predictors in multivariate analysis (RFS P = 0.032; OS P = 0.038 for OS). RD TILs added significant prognostic value to multivariate models including RCB class (P < 0.001 for RFS; P = 0.021 for OS). The positive prognostic effect of RD TILs significantly differed by RCB class for RFS (PInt=0.003) and OS (PInt=0.008) with a greater magnitude of positive effect observed for RCB class II than class III. TIL levels in TNBC RD are significantly associated with improved RFS and OS and add further prognostic information to RCB class, particularly in RCB class II.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2− cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC. Significance: The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients. This article is highlighted in the In This Issue feature, p. 2659