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. 2024 Mar 6;10(1):20.
doi: 10.1038/s41523-024-00625-7.

Cell-cycle inhibition and immune microenvironment in breast cancer treated with ribociclib and letrozole or chemotherapy

Tomás Pascual #  1   2   3   4 , Aranzazu Fernandez-Martinez #  1 , Yash Agrawal #  1 , Adam D Pfefferle  1 , Nuria Chic  3   4 , Fara Brasó-Maristany  4 , Blanca Gonzàlez-Farré  2   4   5 , Laia Paré  2   3   4 , Guillermo Villacampa  2 , Cristina Saura  2   6   7 , Cristina Hernando  8   9 , Montserrat Muñoz  2   3   4 , Patricia Galván  4 , Xavier Gonzàlez-Farré  2   10 , Mafalda Oliveira  2   6   7 , Miguel Gil-Gil  11   12 , Eva Ciruelos  2   13   14 , Patricia Villagrasa  2 , Joaquín Gavilá  2   15 , Aleix Prat  16   17   18   19   20 , Charles M Perou  1
Affiliations

Cell-cycle inhibition and immune microenvironment in breast cancer treated with ribociclib and letrozole or chemotherapy

Tomás Pascual et al. NPJ Breast Cancer. .

Abstract

In this study, we performed genomic analyses of cell cycle and tumor microenvironment changes during and after ribociclib and letrozole or chemotherapy in the CORALLEEN trial. 106 women with untreated PAM50-defined Luminal B early breast cancers were randomly assigned to receive neoadjuvant ribociclib and letrozole or standard-of-care chemotherapy. Ki67 immunohistochemistry, tumor-infiltrating lymphocytes quantification, and RNA sequencing were obtained from tissue biopsies pre-treatment, on day 14 of treatment, and tumor specimens from surgical resection. Results showed that at surgery, Ki67 and the PAM50 proliferation scores were lower after ribociclib compared to chemotherapy. However, consistent reactivation of tumor cell proliferation from day 14 to surgery was only observed in the ribociclib arm. In tumors with complete cell cycle arrest (CCCA) at surgery, PAM50 proliferation scores were lower in the ribociclib arm compared to chemotherapy (p < 0.001), whereas the opposite was observed with tumor cellularity (p = 0.002). Gene expression signatures (GES) associated with antigen-presenting cells (APCs) and innate immune system activity showed increased expression post-chemotherapy but decreased expression post-ribociclib. Interferon-associated GES had decreased expression with CCCA and increased expression with non-CCCA. Our findings suggest that while both treatment strategies decreased proliferation, the depth and the patterns over time differed by treatment arm. Immunologically, ribociclib was associated with downregulated GES associated with APCs and the innate immune system in Luminal B tumors, contrary to existing preclinical data. Further studies are needed to understand the effect of CDK4/6 inhibition on the tumor cells and microenvironment, an effect which may vary according to tumor subtypes.

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Conflict of interest statement

T.P. reports having received Fees for Non-CME Services Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus) from Astra Zeneca, Consulting Fees (e.g., advisory boards) from Novartis, and Fees for Non-CME Services Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus) from Pfizer. L.P. is an employee of Reveal Genomics and has a patent (EP 20 382 679.7—in vitro method for the prognosis of patients suffering from HER2-positive breast cancer). G.V. has received payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing, or educational events from MSD, GSK, Pierre Fabre, and Pfizer and has participated on a data safety monitoring board or advisory board for AstraZeneca. C.S. has served as consultant, participated in advisory boards or received travel grants from AstraZeneca, AX’s Consulting, Byondis BV, Celgene, Daiichi Sankyo, Eisai, F. Hoffmann - La Roche Ltd, Exact Sciences, Exeter Pharma, Genomic Health, Merck, Sharp and Dhome España S.A., Novartis, Odonate Therapeutics, Pfizer, Philips Healthwork, Pierre Fabre, Pint Pharma, prIME Oncology, Puma Biotechnology, Seagen, Synthon, Sanofi Aventis and Zymeworks. M.M. declares the following competing interests: expert testimony honoraria from Novartis, Roche, and Eisai; advisory board honoraria from Pierre Fabre, and Seagen; and conference travel grants from Roche, Pfizer, Gilead and Lilly. X.G. reports honoraria and personal fees from Roche, Novartis and Gilead, Roche, honoraria, travel support, personal fees from AstraZeneca and Pierre Fabre, and personal, travel and congress support from Eli Lilly. M.O. reports financial relationships with AstraZeneca, Guardant Health, Roche, MSD, Pfizer, SeaGen, iTEOS, Eisai, Novartis, Relay Therapeutics and Gilead. E.C. reports personal fees from Roche, personal fees from Lilly, personal fees from Novartis, personal fees from Pfizer, during the conduct of the study. J.G. reports grants and personal fees from NOVARTIS, grants and personal fees from PFIZER, grants and personal fees from ROCHE, outside the submitted work. A.P. has declared personal honoraria from Pfizer, Novartis, Roche, MSD Oncology, Lilly and Daiichi Sankyo, travel, accommodations, and expenses paid by Daiichi Sankyo, research funding from Nanostring Technologies, Roche and Novartis, consulting/advisory role for Nanostring Technologies, Roche, Novartis, Pfizer, Oncolytics Biotech, Amgen, Lilly, MSD, PUMA and Daiichi Sankyo, Inc. outside the submitted work. C.M.P. is an equity stockholder and consultant of BioClassifier LLC; C.M.P. is also listed as an inventor on patent applications for the Breast PAM50 Subtyping assay. The other authors declare no financial or non-financial competing interests.

Figures

Fig. 1
Fig. 1. Comparative antiproliferative impact of ribociclib plus letrozole and multi-agent chemotherapy on luminal B Breast Cancer, as measured by Ki67 expression.
a Individual paired Ki67 expression at baseline and surgery after treatment with ribociclib and letrozole and multi-agent chemotherapy. Colored lines represent individual patient data. b Mean percentage rate of response as determined by complete cell-cycle arrest (CCCA, Ki67 < 2.7%), at week 2 and surgery by treatment arms. c Expression of Ki67 across the three timepoints (Baseline, day 14, surgery) in ribociclib and chemotherapy arms. p-value was obtained after performing ANOVA test. d Individual paired Ki67 expression at baseline, day 14, and surgery. Colored lines represent individual patient data. e Change in Ki67 expression between week 2 and surgery by interval between ribociclib or paclitaxel discontinuation and surgery. Each point represents a patient.
Fig. 2
Fig. 2. Dynamics of gene expression in Luminal B breast cancer: during and post-treatment with ribociclib plus letrozole versus multi-agent chemotherapy.
a Volcano plots of log2 fold change of median gene expression and absolute Score D. Colors point to a significance threshold of FDR < 0.05. Significance was calculated using SAM paired samples analysis. b Bar plot of the D-score of selected signatures in ribociclib and chemotherapy arm between the 3 timepoints. c Venn diagram with signatures upregulated and downregulated (FDR < 0.05) between baseline and day 14 and between baseline and surgery in the ribociclib and chemotherapy arms. d Clusters with signatures selected from ratios between baseline and surgery according to timepoint and arm with a FDR < 0.01 in SAM multiclass analysis. e Clusters with signatures selected from ratios between baseline and surgery according to complete cycle arrest (CCCA) and arm with a FDR < 0.01 in SAM multiclass analysis. f PAM50 proliferation score and percentage of tumor cellularity at baseline and surgery according to CCCA or non- CCCA at surgery. p-value was obtained after performing ANOVA test.
Fig. 3
Fig. 3. Comparative analysis of immune signature expression through supervised gene expression analysis between day 14 samples and surgery tumor specimens, categorized by different treatment arms.
The numerical values correspond to Fold Change, computed using a two-class paired Significance Analysis of Microarrays (SAM), highlighting alterations. Color-coded boxes identify modules exhibiting significant changes with a false discovery rate (FDR) of ≤ 0.05, with upregulation in red and downregulation in green.
Fig. 4
Fig. 4. Differential immune gene expression signatures and correlations in Luminal B breast cancer surgical samples post neoadjuvant treatment.
a Expression of selected signature in surgical samples with complete cycle arrest (CCCA) and non-CCCA in ribociclib arm (left) and chemotherapy arm (right). b Spearman correlation matrix for continuous PAM50 proliferation score and cibersort immune population in surgical samples after neoadjuvant ribociclib and letrozole. c Spearman correlation matrix for continuous PAM50 proliferation score and cibersort immune population in surgical samples after neoadjuvant multi-agent chemotherapy.
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