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. 2024 Apr 24:15:1373497.
doi: 10.3389/fimmu.2024.1373497. eCollection 2024.

Biological effects of intraoperative radiation therapy: histopathological changes and immunomodulation in breast cancer patients

Javier I J Orozco  1 , Betsy J Valdez  1 , Chikako Matsuba  2 , Michael P Simanonok  3 , Miquel Ensenyat-Mendez  4 , Judi Anne B Ramiscal  5 , Matthew P Salomon  2 , Yuki Takasumi  6 , Janie G Grumley  1
Affiliations

Biological effects of intraoperative radiation therapy: histopathological changes and immunomodulation in breast cancer patients

Javier I J Orozco et al. Front Immunol. .

Abstract

Introduction: Intraoperative radiation therapy (IORT) delivers a single accelerated radiation dose to the breast tumor bed during breast-conserving surgery (BCS). The synergistic biologic effects of simultaneous surgery and radiation remain unclear. This study explores the cellular and molecular changes induced by IORT in the tumor microenvironment and its impact on the immune response modulation.

Methods: Patients with hormone receptor (HR)-positive/HER2-negative, ductal carcinoma in situ (DCIS), or early-stage invasive breast carcinoma undergoing BCS with margin re-excision were included. Histopathological evaluation and RNA-sequencing in the re-excision tissue were compared between patients with IORT (n=11) vs. non-IORT (n=11).

Results: Squamous metaplasia with atypia was exclusively identified in IORT specimens (63.6%, p=0.004), mimicking DCIS. We then identified 1,662 differentially expressed genes (875 upregulated and 787 downregulated) between IORT and non-IORT samples. Gene ontology analyses showed that IORT was associated with the enrichment of several immune response pathways, such as inflammatory response, granulocyte activation, and T-cell activation (p<0.001). When only considering normal tissue from both cohorts, IORT was associated with intrinsic apoptotic signaling, response to gamma radiation, and positive regulation of programmed cell death (p<0.001). Using the xCell algorithm, we inferred a higher abundance of γδ T-cells, dendritic cells, and monocytes in the IORT samples.

Conclusion: IORT induces histological changes, including squamous metaplasia with atypia, and elicits molecular alterations associated with immune response and intrinsic apoptotic pathways. The increased abundance of immune-related components in breast tissue exposed to IORT suggests a potential shift towards active immunogenicity, particularly immune-desert tumors like HR-positive/HER2-negative breast cancer.

Keywords: breast neoplasms; immune response; intraoperative radiation therapy-IORT; squamous metaplasia; tumor microenvironment.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Histopathological changes after IORT. (A) Barplots of SM and SMwA in patients with and without IORT. Fisher’s exact test **p=0.004. (B) Distribution of SM and SMwA according to extension as focal (<20% of the slide), multifocal (20-80% of the slide), and diffuse (>80% of the slide). (C) Representative staining of DCIS (left panel) and SMwA (right panel) with differential expression of immunohistochemistry markers (ER and p63). IORT, intraoperative radiation therapy; SM, squamous metaplasia; SMwA, squamous metaplasia with atypia; DCIS, ductal carcinoma in situ; H&E, hematoxylin and eosin; ER, estrogen receptor.
Figure 2
Figure 2
Transcriptomic changes after IORT. Heatmap representing the hierarchical clustering (A) and principal component analysis (PCA) (B) using the 1,000 most variable genes. (C) Volcano plot showing the differentially expressed genes (DEG) between IORT and non-IORT samples (FDR<0.1).
Figure 3
Figure 3
Pathway analyses of gene ontology biological processes. Barplots and network plots showing upregulated gene enrichment pathways using the DEGs for gene ontology (GO) biological process including all tissue types (IORT=21, non-IORT=16) in panels (A, B), and only normal breast tissue (IORT=6, non-IORT=11) in panels (C, D), respectively. Network plots only show significantly upregulated pathways (in green) in IORT samples. Two pathways (nodes) are connected if they share 20% or more genes. Darker nodes indicate the lower adjusted p-value, a larger size of the circle indicates a larger number of genes found in the pathway, and thicker edges represent more overlapped genes.
Figure 4
Figure 4
Cell type enrichment in the tumor microenvironment. Boxplots showing differences between non-IORT and IORT cohorts in immune cell enrichment (A) and immune and stroma scores (B), as determined by the xCell digital portrayal algorithm from TIMER2.0. T-test with Bonferroni corrected values; **p < 0.05, *N.S., non-significant (p = 0.07).
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