When Both Tumour-Infiltrating Lymphocytes and Gene Expression are Available, the Prognostic and Predictive Value of RNA Sequencing–Based Immune Signatures is Superior

In the CALGB 40601 and PAMELA studies, both the proportion of tumour-infiltrating lymphocytes (TILs) and the multiple immune-related gene expression signatures (iGESs) were significantly associated with pathologic complete response (pCR). Moreover, in the CALGB 40601 study, several immune signatures were also associated with event-free survival (EFS) in univariable and multivariable Cox analyses that included clinical factors and intrinsic subtype, whereas TILs were not significantly associated with outcome. B-cell signatures outperformed TILs and T-cell signatures for pCR and EFS. Combining TILs and iGESs did not provide additional prognostic information. These results are especially relevant in early HER2-positive breast cancer, in which multiple studies focus on developing prognostic tools combining tumour and immune cell biomarkers to guide treatment escalation and de-escalation according to Dr. Lisa A. Carey of the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, US and colleagues who published the findings from a correlative analysis on 5 January 2023 in JAMA Oncology.

The authors wrote in the background that there are several methods to assess intratumour immune activation. The percentage of TILs that infiltrate the breast tumour is positively prognostic in patients with early HER2-positive breast cancer treated with anti-HER2 therapies in multiple scenarios. Other than TILs, immune activation can also be measured by gene expression. In patients with early HER2-positive breast cancer treated in the neoadjuvant setting, iGESs are associated with higher pCR rates and prolonged survival. Specifically, the IgG signature has previously shown strong and independent prognostic value across many studies. However, the comparative prognostic ability of these different means of measuring immune activation has not been well examined.

In this retrospective predictive and prognostic study, the investigators tested which biomarker, or combination of biomarkers, is the most powerful for response and survival in 2 independent clinical trials, CALGB 40601 and PAMELA, respectively. The CALGB is now part of the Alliance for Clinical Trials in Oncology. In the CALGB 40601 study, 305 patients were randomly assigned to weekly paclitaxel with trastuzumab, lapatinib, or both for 16 weeks. The primary endpoint was pCR, with a secondary endpoint of EFS. In the PAMELA study, 151 patients received neoadjuvant treatment with trastuzumab and lapatinib for 18 weeks. The primary endpoint was the ability of the HER2-enriched subtype to predict pCR. The studies were conducted from October 2013 to November 2015 (PAMELA) and from December 2008 to February 2012 (CALGB 40601). Data analyses were performed from 1 June 2020 to 1 January 2022.

Immune-related gene expression profiling by RNA sequencing and TILs were assessed on 230 CALGB 40601 study pretreatment tumours and 138 PAMELA study pretreatment tumours. The association of these biomarkers with pCR in CALGB 40601 and PAMELA and EFS in CALGB 40601 was studied by logistic regression and Cox analyses.

The median age of the patients was 50 years and 305 (100%) were women. Of 202 immune signatures tested, 166 (82.2%) were significantly correlated with TILs. In both studies combined, TILs were significantly associated with pCR (odds ratio 1.01; 95% confidence interval [CI] 1.01-1.02; p = 0.02).

In addition to TILs, 36 immune signatures were significantly associated with higher pCR rates. Seven of these signatures outperformed TILs for predicting pCR, 6 of which were B-cell related.

In a multivariable Cox model adjusted for clinicopathologic factors, including PAM50 intrinsic tumour subtype, the IgG signature, but not TILs, was independently associated with EFS (IgG signature–adjusted hazard ratio [HR] 0.63; 95% CI 0.42-0.93; p = 0.02; TIL-adjusted HR 1.00; 95% CI 0.98-1.02; p = 0.99).

The authors concluded that this study supports that measurement of immune activation, either by TIL measurement or by immune-related gene expression profiling, is predictive of treatment response and that immune-related gene expression is prognostic. In the presence of both immune biomarker types, iGESs, especially B-cell–related signatures, outperform TILs for both pCR and prognosis, and the combination of both biomarkers does not yield improved prognostic value. These results highlight the essential role of B cells in antitumour immunity and suggest that B-cell immune-related gene expression provides valuable prognostic information for treatment escalation and de-escalation in patients with early HER2-positive breast cancer.

Research reported in this publication was supported by the US National Cancer Institute of the National Institutes of Health awards. The research was also funded by The Breast Cancer Research Foundation, Susan G. Komen, Fundación SEOM, and Fundación Científica Asociación Española Contra el Cáncer.


Fernandez-Martinez A, Pascual T, Singh B, et al. Prognostic and Predictive Value of Immune-Related Gene Expression Signatures vs Tumor-Infiltrating Lymphocytes in Early-Stage ERBB2/HER2-Positive Breast Cancer A Correlative Analysis of the CALGB 40601 and PAMELA Trials. JAMA Oncology; Published online 5 January 2023. doi:10.1001/jamaoncol.2022.6288