The CD-ROM disc containing supplementary material is kept in the cardboard box in the Systems Office.The heterogeneity of breast cancer (disease stage and phenotype) makes it
challenging to differentiate between each subtype; luminal A, luminal B, HER2,
basal-like and claudin-low, on the basis of a single gene or protein. Therefore,
a collection of markers is required that can serve as a signature for diagnosing
different types of breast cancer. New developments in proteomics have
provided the opportunity to look at phenotype-specific breast cancer cell lines
and stage-specific liquid biopsies (nipple aspirate fluid [NAF], plasma samples)
to identify disease and phenotype specific signature.
An 8-plex iTRAQ quantification strategy was employed to compare proteomic
profiles of a range of breast cancer and ‘normal-like’ cell lines with primary
breast epithelial cells. From this, 2467 proteins were identified on Orbitrap
Fusion and Ultraflex II, of which 1430 were common. Matched pairs of NAF
samples from four patients with different stages of breast cancer, were analysed
by SCX-LC-MS and a total of 1990 unique gene products were identified. More
than double the number of proteins previously published data, were detected in
NAF, including 300 not detected in plasma. The NAF from the diseased patients
have 138 potential phenotype biomarkers that were significantly changed
compared to the healthy volunteer (7 for luminal A, 9 for luminal B, 11 for HER2,
14 for basal-like and 52 for claudin-low type). The average coefficient of
variation for triplicate analyses by multiple reaction monitoring mass
spectrometry (MRM-MS), was 9% in cell lines, 17 % in tissue biopsies, 22% in
serum samples and 24% in NAF samples.
Overall, the results provide a strong paradigm to develop a clinical assay based
on proteomic changes in NAF samples for the early detection of breast cancer
supplementary to established mammography programmes.The supplementary material submitted with the thesis is not available online
