Purpose:

Identifying biomarkers of radiotherapy (RT) response is important for optimizing the treatment of early breast cancer. In this study, we tested the interaction between endothelial cell (EC) expression of phospho-Tyr397-FAK (pY397-FAK) and adjuvant-RT on clinical outcomes after breast-conserving surgery (BCS) within a randomized study. Preclinical data suggest an enhanced effect of RT on low EC_pY397-FAK expression.

Experimental Design:

We analyzed tissue microarrays from the Swedish Breast Cancer Group 91 Radiotherapy (stage I–II, lymph node–negative) breast cancer cohort, consisting of 1,178 patients randomly assigned to receive either BCS alone or BCS plus adjuvant-RT. Tissue microarray sections were immunostained for pY397-FAK, CD31, α-smooth muscle actin, and pan-cytokeratin. HALO analysis scored mean pY397-FAK intensity in CD31+ ECs, pan-cytokeratin–positive tumor epithelial cells, and α-smooth muscle actin + mural/stromal cells per core. For 822 patients, multivariable Cox regression analysis was performed for the primary and secondary 5-year endpoints, locoregional recurrence and all recurrence, respectively, as dependent variables and RT and EC_pY397-FAK as independent variables.

Results:

EC_pY397-FAK expression was not predictive for the primary endpoint locoregional recurrence (P = 0.098), but the direction of the RT effect was in line with preclinical findings. For the secondary endpoint all recurrence, there was a significant interaction (P = 0.026) between EC_pY397-FAK and RT. Without RT, higher EC_pY397-FAK expression resulted in a lower risk for all recurrence (HR = 0.74 per SD; 95% confidence interval = 0.57–0.96; P = 0.026).

Conclusions:

Within the first 5 years following BCS, patients with low EC_pY397-FAK expression derive greater benefit from RT than patients with high EC_pY397-FAK expression. However, without RT, low EC_pY397-FAK expression is associated with a higher risk of recurrence.

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©2025 The Authors; Published by the American Association for Cancer Research
2025
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
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Supplementary data

Supplementary Figure S1

Graphical summary of the Cell DIVE and HALO analysis methods used for measuring pY397-FAK intensity patient tissue microarrays (TMAs). CAFs, Cancer-associated fibroblasts; H&Es, Haemotoxylin and Eosin; ROI, Region-of-interest; QC, Quality control; panCK, pan-cytokeratin; ααSMA, alpha-smooth muscle actin; TC, tumour cell; EC, endothelial cell; pY397-FAK, phospho-FAK(Tyr397). (Created in BioRender. Drake, R. (2025) https://BioRender.com/f89g292).

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Supplementary Figure S2

Distribution of average pY397-FAK intensity scores. Data are given per patient, from primary tumour cores only, in the CD31+ endothelial cells (A), panCK+ tumour cells (B) and ααSMA+ region (C), and average CD31 positive-stained blood vessel area (µm2) across patient primary tumours (D).

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Supplementary Figure S3

Immunofluorescence staining for pY397-FAK, CD31, PanCK and αSMA. Representative immunofluorescence images of a patient in the highest and lowest quartiles of pY397-FAK intensity, in the CD31-positively stained endothelial cells (EC_ pY397-FAK) (A), panCK-positively stained epithelial tumour cells (TC_ pY397-FAK) (B) and ααSMA-positively stained mural and stromal cells (αSMA_ pY397-FAK) (C). Lowest panels represent high power regions of images in the row above denoted by the dotted boxes.

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Supplementary Figure S4

Spearman’s rank correlations (A) Spearman’s rank correlations of patient characteristics and pY397-FAK scores for EC, TC and ααSMA region. Upper figures, r-values; lower figures p-values. (B) No correlation between EC_ pY397-FAK and CD31+ area, spearman’s rank (r = 0.05556, P = 0.1099) ααSMA, alpha-smooth muscle actin; EC, endothelial cell; ER, estrogen receptor; IHC, Immunohistochemistry; pY397-FAK, phosphorylated-FAK(Y397); TILs, tumour infiltrating lymphocytes; TC, tumour cell.

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Supplementary Table S1

Table of Antibodies. List of primary, conjugated and secondary antibodies stained for in each round: CD31, α-SMA, Pan-CK and pY397-FAK.

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Supplementary Table S2

Median and interquartile ranges for each pY397-FAK variable as distributed across the NoRT and RT arms

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Supplementary Table S3

Representativeness of Study participants

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Supplementary Table S4

Outcome is locoregional recurrence for TC_pY397-FAK, 5-year follow-up.

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Supplementary Table S5

Outcome is all recurrence for TC_pY397-FAK, 5-year follow-up.

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Supplementary Table S6

Outcome is locoregional recurrence for αSMA_pY397-FAK, 5-year follow-up

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Supplementary Table S7

Outcome is all recurrence for αSMA_pY397-FAK, 5-year follow-up.

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