Functional ex vivo DNA fibre assay to measure replication dynamics in breast cancer tissue

Mengting Chen, Nathalie van den Tempel, Arkajyoti Bhattacharya, Shibo Yu, Bea Rutgers, Rudolf S.N. Fehrmann, Sander de Haas, Bert van der Vegt, Marcel A.T.M. van Vugt*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = −0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = −0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs.

Original languageEnglish
Pages (from-to)90-100
Number of pages11
JournalJournal of Pathology
Volume264
Issue number1
DOIs
Publication statusPublished - Sept-2024

Keywords

  • breast cancer
  • copy number variations
  • Cyclin E1
  • DNA fibre assay
  • genomic instability
  • replication stress

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