Immunology Biomarkers Taking Shape in Breast Cancer

The search for immune system biomarkers that could prove clinically useful in treating patients with breast cancer is yielding promising results, particularly in triple-negative subtypes.

Elias Obeid, MD, MPH

The search for immune system biomarkers that could prove clinically useful in treating patients with breast cancer is yielding promising results, particularly in triple-negative subtypes, according to a recent review of research findings from the 2015 San Antonio Breast Cancer Symposium (SABCS).

Abstracts exploring tumor-infiltrating lymphocytes (TILs) and CD8-positive T cells as prognostic markers were among the immunology highlights presented at SABCS, said Elias Obeid, MD, MPH, an assistant professor in the departments of Clinical Genetics and Medical Oncology at Fox Chase Cancer Center.

Obeid spoke during an annual SABCS review that Fox Chase organized in January for oncology specialists and healthcare professionals throughout the Philadelphia region. Lori J. Goldstein, MD, director of the Naomi and Phil Lippincott Breast Evaluation Center and deputy associate director of Clinical Research at Fox Chase, served as program chair for the event, which also featured researchers from the Hospital of the University of Pennsylvania.

The focus on immunological biomarkers in breast cancer has been growing in recent years as evidence builds about the prognostic and potentially predictive value of TILs in triple-negative breast cancer (TNBC) and HER2-overexpressing breast cancer. At the same time, monoclonal antibodies that target the PD-1/PD-L1 immune checkpoints are being investigated in breast cancer following their success in melanoma and non—small cell lung cancer, intensifying the importance of identifying immune system biomarkers.

Last year, the International TILs Working Group of breast cancer researchers called for a standardized methodology for evaluating TILs that could be employed in research and in clinical trials.1 The group also noted that the presence of specific subsets of immune cells, notably cytotoxic CD8-positive T cells, have been correlated with clinical responses to therapy in breast cancer.1

For TILs, the Working Group said that values should be reported for the stromal compartment; that is, as the percentage of stromal TILs in the area of stromal issue. They did not make a recommendation on a clinically relevant TIL threshold.

Tumor-Infiltrating Lymphocytes

In his presentation, Obeid recapped key points and findings from 3 SABCS abstracts concerning these immunology biomarkers.In one study, Loi et al2 analyzed pooled data from 991 patients with early-stage TNBC treated with adjuvant chemotherapy during 6 randomized clinical trials. Sixty-two percent of the patients had been treated with anthracycline plus taxane chemotherapy, while 38% were treated with an anthracycline-based regimen alone.

The researchers were seeking to “achieve a robust understanding” of the prognostic value of TILs in this disease setting; secondary objectives included developing a model to determine the value of TILs as an added variable to standard clinicopathological factors.

The investigators measured intratumoral TILs, defined as those lymphocytes with direct contact to tumor cells, and stromal TILs, which are between the tumor cells but within the tumor stroma. Overall, the average value of stromal TILs was 20%, and 90% of patients had at least 1% stromal TILs in the samples tested, investigators indicated.

The study showed that the presence of stromal TILs was associated with a 16% reduction in both invasive disease-free survival (DFS) events and in the risk of death (HR, 0.84). On multivariate analysis, researchers found that stromal TILs were significantly associated with invasive and distant DFS and with overall survival.

However, intratumoral TILs did not significantly correlate with outcomes, noted Obeid. Moreover, patient age and nodal status were not significantly associated with TILs but “larger tumor size had lower infiltration with lymphocytes,” Obeid said.

In conclusion, Obeid said, patients with node-negative TNBC and stromal TILs ≥20% have excellent 5-year distant DFS outcomes, making it possible to develop a model to predict risk of distant recurrence incorporating TILs.

“For every 10% increase in stromal infiltration in the tumor, there was about a 12% relative risk reduction adjusted for different variables,” Obeid noted. “Clinical pathologic factors in addition to stromal infiltrating tumor lymphocytes seem to be enough in this model.”

In their abstract, researchers concluded that stromal TILs should be “strongly considered” as a stratification factor in future clinical trials for patients with TNBC. “Given the important prognostic role of preexisting immunity, patients with TNBC are rational candidates for immunotherapy clinical trials,” they added.

Immune Signature Emerges

“Definitely, this is promising,” Obeid commented. “It needs validation in larger datasets.”In the BEATRICE study, investigators evaluated whether the addition of bevacizumab to standard adjuvant chemotherapy would improve outcomes for patients with resected, invasive, early TNBC. The randomized phase III study enrolled nearly 2600 patients.

Obeid focused on an exploratory biomarker analysis of tumor specimens from 1105 participants in the BEATRICE trial.3 The formalin-fixed, paraffin-embedded tumor samples were analyzed using hematoxylin and eosin (H&E) staining for a number of biomarkers including TILs, a CD8 effector T-cell signature, an immune checkpoint signature, B cells, and macrophages.

The presence of TILs greater than the median 7% level was associated with better prognosis (HR, 0.69) while higher levels of stromal cells greater than 25% were linked to a worse prognosis (HR, 1.47) in terms of invasive DFS, Obeid said.

The CD8 effector T-cell signature, composed of 6 T-cell associated genes (CD8A, GZMA, GZMB, IFNG, EOMES, PRF1) positively correlated with invasive DFS when present at higher levels (HR, 0.43). Similarly, an immune checkpoint signature that measured PD-L1, PD-L2, and IDO1 expression was associated with improved DFS prognosis (HR, 0.55).

Obeid said B-cell levels were “weakly” associated with a good prognosis (HR, 0.72). However, a stroma signature composed of cancer-associated fibroblasts, ESM-1, and Notch signaling was associated with worse prognosis (HR, 1.44, 1.53, and 1.81, respectively), he said.

Lymphocytes in Lobular Cancers

Molinero et al reported that the finding for the immune checkpoint signature “suggests equilibrium between cytotoxic T cells and their inhibitors” in early TNBC, thus building evidence for evaluating those immunotherapy agents in the subtype.For the third immunology biomarker abstract, Obeid reviewed what he described as a “unique” study of TIL characteristics in invasive lobular carcinoma (ILC), which represents an estimated 5% to 15% of all breast cancer cases. Desmedt et al said their research represents the largest-ever TIL analysis in the subtype.4

In the study, investigators focused on 555 ER-positive, HER2-negative tumor samples. The median value of stromal TILs, assessed via H&E staining, was 5%, with an interquartile range of 5% to 10%; only 9% of the samples had >20% stromal TILs.

Obeid said the analysis showed that most of the ILC samples had a lower distribution of TILs than a small number of invasive ductal tumors examined (n = 37), “tilting more toward the higher TILs in invasive ductal versus invasive lobular.”

When the ILC samples were compared with clinicopathologic factors, “higher TILs in invasive lobular carcinoma correlated with a more aggressive phenotype,” Obeid said. These factors included younger age at diagnosis, axillary lymph node involvement, high proliferative tumors as assessed by Ki67, and mixed nonclassic ILBC subtypes, the abstract indicated.

The researchers also found greater numbers of TILs were associated with ARID1A, BRCA2, KMT2C, and TP53 gene mutations and with loss in chromosomes chr3p21.31 and chr8q24.23 (PTK2). “There is an association with some of the gene mutations in the tumor so this is not germline, this is somatic,” noted Obeid.

However, the signals were different when it came to correlating the prognostic value of TILs with breast cancer—free survival after 12 years’ follow-up among patients whose tumors were analyzed. “In a univariate analysis, tumors with the highest content of tumor-infiltrating lymphocytes seem to have done worse and that was significant, but in a multivariate model this actually failed to show the same outcome,” said Obeid.


  1. Salgado R, Denkert C, Demaria S, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol. 2015;26(2):259-271.
  2. Loi S, Drubay D, Adams S, et al. Pooled individual patient data analysis of stromal tumor infiltrating lymphocytes in primary triple negative breast cancer treated with anthracycline-based chemotherapy. Presented at: 2015 San Antonio Breast Cancer Symposium; December 9-12, 2015; San Antonio, TX. Abstract S1-03.
  3. Molinero L, Yu J, Li C, et al. Analysis of molecular prognostic factors associated with tumor immune and stromal microenvironment in BEATRICE, an open-label phase 3 trial in early triple-negative breast cancer (eTNBC). Presented at: 2015 San Antonio Breast Cancer Symposium; December 9-12, 2015; San Antonio, TX. Abstract S1-01.
  4. Desmedt C, Salgado R, Buisseret L, et al. Lymphocytic infiltration in invasive lobular breast cancer. Presented at: 2015 San Antonio Breast Cancer Symposium; December 9-12, 2015; San Antonio, TX. Abstract S1-02.