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Oncoproteomic applications for detection of breast cancer. Proteomic profiling of breast cancer models and biopsies
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 ānormal-likeā 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
High-Throughput Proteomic Profiling of Nipple Aspirate Fluid from Breast Cancer Patients Compared with Non-Cancer Controls: A Step Closer to Clinical Feasibility.
BACKGROUND: Early detection of breast cancer (BC) is critical for increasing survival rates. However, current imaging approaches can provide ambiguous results, requiring invasive tissue biopsy for a definitive diagnosis. Multi-dimensional mass spectrometric analysis has highlighted the invaluable potential of nipple aspirate fluid (NAF) as a non-invasive source of early detection biomarkers, by identifying a multitude of proteins representative of the changing breast microenvironment. However, technical challenges with biomarker validation in large cohorts remain due to low sample throughput, impeding progress towards clinical utility. Rather, by employing a high-throughput method, that is more practicable for clinical utility, perturbations of the most abundant NAF proteins in BC patients compared with non-cancer (NC) controls could be monitored and validated in larger groups. METHOD: We characterized matched NAF pairs from BC (n = 9) and NC (n = 4) volunteers, using a rapid one dimensional liquid chromatography-mass spectrometry (1D LC-MS/MS) approach. RESULTS: Overall, 198 proteins were relatively quantified, of which 40 were significantly differentiated in BC samples, compared with NC (p ā¤ 0.05), with 26 upregulated and 14 downregulated. An imbalance in immune response and proteins regulating cell growth, maintenance and communication were identified. CONCLUSIONS: Our findings show 1D LC-MS/MS can quantify changes reflected in the NAF proteome associated with breast cancer development
Evaluation of nipple aspirate fluid as a diagnostic tool for early detection of breast cancer
YesThere has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds
of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancerrelated
deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style
as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast
cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography
plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive
in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging
approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection
methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal
women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease
in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The
emergence of āomicsā strategies over the last 20 years, enabling the characterisation and understanding of breast cancer
at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and
serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully
translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies,
such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively
for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection
kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for
routine breast health monitoring. The current status of established and newly emerging imaging techniques for early
detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those
applied to high-risk, premenopausal women, will be reviewed.Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA, and co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation projects Ī„ĪĪĪĪ/ĪĪĪĪ£/0311(ĪĪĪ/07) and NEKYP/0311/17
A proteomic investigation to discover candidate proteins involved in novel mechanisms of 5-fluorouracil resistance in colorectal cancer
YesOne of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development
of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some
resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is
still unknown. Here, a proteomics approach is taken towards identification of candidate proteins
using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable
isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently
passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with
2D liquid chromatography and Orbitrap Fusionā¢ Tribridā¢ Mass Spectrometry analysis. Among
3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found
up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total
of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC
oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44
and CD63, two known drug resistance mediators with elevated proteomics expression results. The
information revealed by the sensitivity of this method warrants it as an important tool for elaborating
the complexity of acquired drug resistance in CRC.Sadr ul-Shaheed and the University of Bradford Proteomics Facility were supported by Yorkshire Cancer Research, UK (Cancer Medicine Discovery II, grant B381PA)
Identification of Stage-Specific Breast Markers using Quantitative Proteomics
YesMatched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships
between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.Cyprus Research Promotion Foundation, Yorkshire Cancer Researc
High-Throughput Proteomic Profiling of Nipple Aspirate Fluid from Breast Cancer Patients Compared with Non-Cancer Controls: A Step Closer to Clinical Feasibility
Background: Early detection of breast cancer (BC) is critical for increasing survival rates. However, current imaging approaches can provide ambiguous results, requiring invasive tissue biopsy for a definitive diagnosis. Multi-dimensional mass spectrometric analysis has highlighted the invaluable potential of nipple aspirate fluid (NAF) as a non-invasive source of early detection biomarkers, by identifying a multitude of proteins representative of the changing breast microenvironment. However, technical challenges with biomarker validation in large cohorts remain due to low sample throughput, impeding progress towards clinical utility. Rather, by employing a high-throughput method, that is more practicable for clinical utility, perturbations of the most abundant NAF proteins in BC patients compared with non-cancer (NC) controls could be monitored and validated in larger groups. Method: We characterized matched NAF pairs from BC (n = 9) and NC (n = 4) volunteers, using a rapid one dimensional liquid chromatography-mass spectrometry (1D LC-MS/MS) approach. Results: Overall, 198 proteins were relatively quantified, of which 40 were significantly differentiated in BC samples, compared with NC (p ā¤ 0.05), with 26 upregulated and 14 downregulated. An imbalance in immune response and proteins regulating cell growth, maintenance and communication were identified. Conclusions: Our findings show 1D LC-MS/MS can quantify changes reflected in the NAF proteome associated with breast cancer development
Rational Development of Novel Activity Probes for the Analysis of Human Cytochromes P450
Perfusate Proteomes Provide Biological Insight Into Oxygenated Versus Standard Hypothermic Machine Perfusion in Kidney Transplantation
Objective: To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate samples collected during a phase 3 randomized double-blind paired clinical trial of hypothermic machine perfusion with and without oxygen (COMPARE). Background: Despite the clinical benefits of novel perfusion technologies aiming to better preserve donor organs, biological processes that may be altered during perfusion have remained largely unexplored. The collection of serial perfusate samples during the COMPARE clinical trial provided a unique resource to study perfusate proteomic profiles, with the hypothesis that in-depth profiling may reveal biologically meaningful information on how donor kidneys benefit from this intervention. Methods: Multiplexed liquid chromatography-tandem mass spectrometry was used to obtain a proteome profile of 210 perfusate samples. Partial least squares discriminant analysis and multivariate analysis involving clinical and perfusion parameters were used to identify associations between profiles and clinical outcomes. Results: Identification and quantitation of 1716 proteins indicated that proteins released during perfusion originate from the kidney tissue and blood, with blood-based proteins being the majority. Data show that the overall hypothermic machine perfusion duration is associated with increasing levels of a subgroup of proteins. Notably, high-density lipoprotein and complement cascade proteins are associated with 12-month outcomes, and blood-derived proteins are enriched in the perfusate of kidneys that developed acute rejection. Conclusions: Perfusate profiling by mass spectrometry was informative and revealed proteomic changes that are biologically meaningful and, in part, explain the clinical observations of the COMPARE trial.</p