The role of intra-tumoural heterogeneity in resistance to neoadjuvant chemotherapy in breast cancer

Breast cancer is a heterogeneous disease and accumulating evidence suggests that treatment failure may be driven by intra-tumour heterogeneity (ITH). Utilising the current protocol for neoadjuvant (pre-surgery) chemotherapy (NAC) provides the opportunity to study molecular genetic changes between pre- and post-therapy by assessing pre-therapy biopsies and post-therapy surgical resections. Whole exome sequencing was performed on matched pre- and post-treatment cancer cells from 6 patients with oestrogen receptor positive breast cancers that showed partial responses to the chemotherapeutic combination epirubicin/cyclophosphamide. Data analysis was performed to determine differences in genetic aberrations between pre- and post-NAC, and in particular to identify evidence of consistent selection by therapy of aberrations that therefore may define chemotherapy resistance or sensitivity. There were extensive differences in the range of genetic aberrations between pre- and post-NAC. 48 genes were identified for further study based on evidence of mutations conferring a selective advantage or disadvantage during chemotherapeutic response. The relevance of these was screened using siRNA knock-down and assessment of response to epirubicin using cell viability assays in vitro. Two genes were taken forward. Potential loss-of-function mutations in MUC17 were selected against during therapy in patients, and in accordance with this MUC17 knock-down was associated with increased sensitivity in vitro. Potential loss-of-function mutations in PCNX1 were selected for during therapy in patients, and in accordance with this PCNX1 knock-down was associated with resistance. Further work was performed to investigate mechanisms by which these genes modify chemotherapy response, by examining drug loading and ABC transporter expression levels. Data indicate that both genes impact on drug loading, potentially through modulating ABC transporter expression. Also, MUC17 or PCNX1 protein levels were tested as prognostic and predictive markers for breast cancer clinical outcomes using tissue taken from cohorts of patients who received adjuvant chemotherapy or neoadjuvant chemotherapy. Kaplan-Meier survival analyses revealed that low MUC17 expression after neoadjuvant chemotherapy was significantly associated with longer disease free survival, which was in agreement with the selection of MUC17 mutations seen after therapy in the initial patient group, and with the in vitro siRNA findings concerning drug sensitivity. I concluded that MUC17 and PCNX1 are potential markers of response to chemotherapy in breast cancer, and that therapeutic modulation of their activities could enhance chemotherapy responses.