Dr Jason Adhikaree - Identifying drivers of metastatic risk and treatment resistance in triple negative breast cancer undergoing neoadjuvant chemo-immunotherapy treatment.
Lay summary
Background: The survival for patients with locally advanced triple negative breast cancer remains lower than other subgroups due to treatment resistance and early metastasis. Tumours shed parts of itself called extracellular vesicles (EV) which carry tumour specific messengers to prepare for metastatic implantation in an organ. Not only do EVs establish metastasis, their persistence during treatment, we hypothesize, they hold the potential to understand treatment resistance and mechanisms of disease relapse.
Aims: We aim to isolate EV from 3 triple-negative banked patient derived cell lines and analyse the size, shape and internal content (RNA and proteins).We then aim to take serial blood samples from 10 patients at baseline and at 12 weeks into treatment (after 4 cycles) of neoadjuvant chemoimmunotherapy to assess for persistent EV post treatment. We will use advanced sequencing technologies to analyse the content of the EV and computer programmes to quantify the largest changes in the internal content pre- and post-treatment.
Impact: The triple negative breast cancer patient group remains a high unmet need for improved survival. Immunotherapy alongside chemotherapy has beenthe major advance in this field for both early-stage disease as well as advanced stage where it offers long-term survival to a few. However, many patients become resistance to chemo-immunotherapy which remains an unanswered dilemma for triple negative breast cancer, and indeed for all high mortality associated cancers. Our hypothesis is that EV present at diagnosis and more importantly, persistent after treatment, hold key information on factors driving relapse and/or disease progression. We aim to discover prognostic signatures warranting higher risk and identify new cancer metastatic and drug resistance pathways. We envisage this research leading to a larger grant for prospective cohort study to validate ourfindings. Ultimately, we aim to improve treatments for triple negativebreast cancer patients.
Scientific summary
Background: Triple negative breast cancer remains a difficult to treat cancer. The overall survival with early-stage disease has significantly improved with the addition of Pembrolizumab to chemotherapy backbones, but 15% still relapse within 3 years[1]. Chemoimmunotherapyalso remains the backbone of treatment in PD-L1 +ve advanced cancers following the Keynote-355 trial which established immunotherapy as the first treatmentto increase survival in this disease[2]. Hence, almost all triple-negative breast cancer patients will be exposed to chemo-immunotherapy. Understanding why patients recur oracquire resistance is vital for the next wave of treatments. Extracellular vesicles (EV) have long been understood as important for cancer metastasis. EV establish a pre-metastatic niche by carrying specific integrins or microRNAs (miRNA) which are organ specific andhas advanced our understanding of mechanisms of metastasis in cancer[3]. In breastcancer there has been an established link with EV and metastatic sites like to the lungsand bones including models of triple negative cancer[4-7]. Recently, in 3 cell lines including 2 triple negative cancers, increased miR-378a-3p and miR-378d levels were seensecreted from cancer cells in exosomes post chemotherapy treatment. The downstream consequence was shown to be activation of the WNT/β-catenin and Notch stem cell associated pathways, thereby inducing chemoresistance[8]. What is not well identified isthose EV which persist in the serum of patients post treatment. We hypothesis that these EV may contain the mechanism for metastatic relapse or treatment resistance.
Aims: 1. To phenotype, enumerate and sequence the EV from patient derived cell lines preand post platinum doublet and immunotherapy exposure2. To phenotype serum EV from patients with triple negative breast cancer pre andpost chemo-immunotherapy treatment to identify persistent EV linked to relapse and progression and correlate with tumour response (complete pathologicalresponse (cPR) vs non-cPR))
Techniques and Methodology: Initially, 3 triple negative breast cancer patient-derived cell line culture models will beinvestigated. Cell lines will be cultured in associated media to 80% confluence and drugged at titrating time points with chemotherapy and immunotherapy at titrated doses. The optimal conditions verified by cell count and visual inspection. EV analysis ofthe appropriate condition (pre or post treatment) pellets will be harnessed and culturedin exosome free media for 48hours. The supernatant will then be harvested andimmediately centrifuged in 100kDa molecular weight cutoff protein concentratorcolumns to a final 500μl volume. EV will then be further purified using 70nM sizeexclusion chromatography columns. Half will be stored for RNA analysis. The resultantsample is then diluted (1:500-1:1000) and the EVs are phenotype and enumerated using Nanoparticle tracking analysis. The stored sample mRNA and miRNA will be extractedfrom a cell pellet using the NucleoSpin RNA Plus Kit (Machery-Nagel) and miRNeasyMini Kit (Qiagen) respectively, according to the manufacturer’s protocol. The sample will be sequence in the University of Nottingham DeepSeq laboratory using NanoporeMinION sequencing device. The raw data will be analysed using DESeq2 bio conductor package to perform differential expression analysis. miRNA target prediction toolsincluding TargetScan and R2 Genomics Analysis and Visualisation Platform will be usedto predict target pathways of significance.For patient samples, blood will be collected in 2xEDTA tubes. The plasma will beharvested by published EV isolation protocols from patient peripheral blood[9] and then handled as described. The data will be correlated to clinical data on the standard of carepathway specifically, histological response and/or radiological response performed bythe NHS teams.
Impact on breast cancer research: The triple negative breast cancer patient group remains a high unmet need for improved survival. Immunotherapy alongside chemotherapy has been the major advance in this field for both early-stage disease as well as advanced stage where it offers long-term survival to a few. However, many patients become resistance to chemo-immunotherapy which remains an unanswered dilemma for triple negativebreast cancer, and indeed for all high mortality associated cancers. Our hypothesis isthat EV present at diagnosis and more importantly, persistent after treatment, hold key information on factors driving relapse and/or disease progression. We aim to discover prognostic signatures warranting higher risk and identify new cancer metastatic and drug resistance pathways. We envisage this research leading to a larger grant for prospective cohort study to validate our findings. Ultimately, we aim to improvetreatments for triple negative breast cancer patients.