Targeted Chemotherapy Using Focused Ultrasound for Liver Tumours

Analytical chemistry (High Performance Liquid Chromatography) for total doxorubicin (including both released and unreleased forms) was performed on section of intratumoral biopsy samples in Good Clinical Practice Laboratory, using a validated assay. Doxorubicin concentration was evaluated in biopsy samples both post-LTLD and post-LTLD+FUS. Tumour samples were not analysed same day and were frozen at -80\^oC for subsequent analysis. Required to evaluate the primary endpoint.

Time frame: Post-intervention sample (Day 1) compared to pre-intervention sample (Day 1)

To satisfy the primary endpoint, a demonstrable two-fold increase in\*, or value exceeding 10μg/g of, the concentration of intra-tumoural doxorubicin at the treated tumour site following FUS-induced mild hyperthermia, was required in at least 50% of evaluable participants. \* As per the a priori protocol design, in Part II the biopsy prior to FUS-induced mild hyperthermia is not performed and therefore the average value for all evaluable tumours receiving intervention in Part I is used as a comparison for the two-fold increase from pre-FUS to post-FUS biopsy.

Time frame: Post-LTLD+FUS sample (Day 1) compared to Post-LTLD sample (Day 1)

Achievement of hyperthermia in the target liver tumour, as determined by real-time thermometry obtained by an indwelling thermometry device. For success, sustained and controlled hyperthermia is required in the target tumour, consequent with drug release (in excess of 39.5\^oC). Real time thermometry plots for each Part I patient are available in the key Lancet Oncology publication, details available in the References section.

Time frame: Real-time thermometry monitoring during intervention (Day 1)

Population: In Part II there was no real-time thermometry and this endpoint only applies to Part I patients.

Adverse Events are listed separately in the subsequent results, but were also specified as a secondary endpoint in the a priori protocol and thus significant events are summarised here. 'Definitely' or 'Probably' related events are included.

Time frame: Up to 30 days post-intervention (Day 1-30)

Adverse Events are listed separately in the subsequent results, but were also specified as a secondary endpoint in the a priori protocol and thus significant events are summarised here. 'Definitely' or 'Probably' related events are included.

Time frame: Up to 30 days post-intervention (Day 1-30)

Post-LTLD+FUS tissue from the targeted liver tumours was obtained by biopsy at the time of the intervention, between 24/03/2015 and 29/03/2017. Cytokeratin-8 (CK-8) is a cell viability marker which if present, demonstrates lack of ablative cell death by any ablative modality, including FUS. Not all histological cell types express CK8, thus if the Post-LTLD+FUS it may either indicate: i) Non-CK8 expression ii) Thermal ablation and consequent cell death. Note there was uncertainty about CK8 expression of individual patient tumours prior to recruitment. In this study if the Post-LTLD+FUS tissue shows specific cellular CK8 cellular staining, then it demonstrates that i) the tumour is CK8+, and, ii) the tumour was not instantaneously thermally ablated and any subsequent cell death is likely due to drug delivery/chemo-ablation. For more information see key TARDOX Lancet Oncology publication and Cytokeratin 8 reference (both detailed in References section).

Time frame: Tissue obtained on day of intervention (Day 1). All CK8 cell viability staining was performed within 2 months of sampling.

Population: 5/6 Part I patients and 4/4 Part II patients had tissue which was analysable for CK-8. The Outcome Measure Data Table demonstrates the number of patients from each arm having CK-8 positive tumour biopsy samples post-LTLD+FUS, indicating lack of thermal ablation (which is consistent with desired hyperthermia rather than undesirable FUS ablation).