TGFβI gene promoter methylation as a biomarker of resistance to anti-HER2 therapies in HER2-positive breast cancer

ENG- HER2 positive breast cancer represents approximately 15-20% of breast cancer and it is associated with higher grade, more aggressive phenotype and worse prognosis. The prognosis of these patients has dramatically changed since the discovery of anti-HER2 therapies. Despite the efficacy of anti-HER2 agents, some patients do not respond to treatment, while others will eventually progress. Understanding the molecular mechanisms of anti-HER2 drug resistance are crucial to identifying patients that fail to respond to the therapy upfront and help design more optimal treatment strategies. Tumorigenesis is a multistep process resulting from the accumulation of genetic alterations but epigenetic alterations also play an important role in cancer development. Tumour biomarkers may be used for determining prognosis and predicting therapeutic response. Taking the previous in vitro results from a preclinical study, our goal was to determine whether the presence of the TGFβI promoter CpG hypermethylation in trastuzumab resistance model also occurs in HER2+ breast cancer patients suggesting a potential biomarker of resistance. This would determine if this could be a factor to discriminate between responders from non-responders and contribute to patient stratification and personalised treatment. Furthermore, analyses of methylation information in international public datasets has been made in order to compare them with our results. Our cohort included 96 patients diagnosed with HER2+ BC treated from 2016 to 2022 in the Girona health care region treated with neoadjuvant chemotherapy followed by trastuzumab +/- pertuzumab. A total of samples from 77 patients could be retrospectively evaluated. Among those, 44 were from responder patients (pretreatment samples) whereas 33 were paired samples from non-responder patients (pretreatment and posttreatment samples). Clinical, histopathological features and survival outcomes were obtained for each patient. Methylation levels in pretreatment samples from responders and non-responders were analysed by bisulfite pyrosequencing and it was also evaluated in posttreatment samples of non-responder patients. A bioinformatic analysis has been carried out with the data available in public data repositories (TCGA and Metabric). Regarding the comparison of epigenetic analyses of the pretreatment methylation levels in responders and non-responders, no statistically significant differences were observed, but results were numerically relevant. Mean methylation levels of the pretreatment and posttreatment samples within the group of non-responders were also compared without statistically significant differences. Promoter hypermethylation of TGFβI as an individual parameter does not allow to differentiate whether an individual will be a responder or a non-responder. From the perspective of clinicopathological factors, ER positivity and Ki67 levels below 20% are predictors of non-response. From public tumour datasets, age has been the only clinical variable associated with methylation