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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 ≤ 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.

The aim of the study is to identify cornulin (CRNN) protein expression associated with advancement of tongue squamous cell carcinoma (TSCC). A comparison of addictive (containing potential carcinogens) versus non-addiction causative agents was expected to allow detection of differences in CRNN expression associated with TSCC. Bespoke tissue microarrays (TMAs) were prepared and immunohistochemistry (IHC) performed to determine the changes in CRNN expression in epithelial cells of node-negative (pN-), node-positive (pN+) TSCC and non-cancer patients’ oral tissues. TMAs were validated by performing IHC on whole diagnostic tissues. Chi-square test or Fisher’s-exact tests were used to establish significant expression differences. Analogous analyses were performed for biomarkers previously associated with TSCC, namely collagen I alpha 2 (COL1A2) and decorin (DCN) to compare the significance of CRNN. Keratinisation and its level (low, extensive) were studied in relation to CRNN so that the extent of squamous differentiation could better be assessed. IHC immunoreactive score (IRS) clustered the patients based on weak/moderate (Low (IRS ≤ +3)) or strong (High (IRS ≥ +4)) expression groups. A low expression was observed in a larger number of patients in control proteins COL1A2 (77.3%), DCN (87.5%) and target protein CRNN (52.3%), respectively. Low CRNN expression was observed in TSCC where nodes were involved (pN+: mean 1.4 ± 2.1) (p = 0.248). Keratinisation (%) was low (0% ≤ 50%) in 42.2% and extensive (1% ≥ 50.0%) in 57.8% patients. In conclusion, our study suggested that Low CRNN expression was associated with grade and lymph node metastasis in TSCC. CRNN expression is independent of addiction, however potentially carcinogenic addictive substances might be aiding in the disease progression.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

Background The aim of this pilot study was to identify proteins associated with advancement of colon cancer (CC). Methods A quantitative proteomics approach was used to determine the global changes in the proteome of primary colon cancer from patients with non-cancer normal colon (NC), non-adenomatous colon polyp (NAP), non-metastatic tumor (CC NM) and metastatic tumor (CC M) tissues, to identify up- and down-regulated proteins. Total protein was extracted from each biopsy, trypsin-digested, iTRAQ-labeled and the resulting peptides separated using strong cation exchange (SCX) and reverse-phase (RP) chromatography on-line to electrospray ionization mass spectrometry (ESI-MS). Results Database searching of the MS/MS data resulted in the identification of 2777 proteins which were clustered into groups associated with disease progression. Proteins which were changed in all disease stages including benign, and hence indicative of the earliest molecular perturbations, were strongly associated with spliceosomal activity, cell cycle division, and stromal and cytoskeleton disruption reflecting increased proliferation and expansion into the surrounding healthy tissue. Those proteins changed in cancer stages but not in benign, were linked to inflammation/immune response, loss of cell adhesion, mitochondrial function and autophagy, demonstrating early evidence of cells within the nutrient-poor solid mass either undergoing cell death or adjusting for survival. Caveolin-1, which decreased and Matrix metalloproteinase-9, which increased through the three disease stages compared to normal tissue, was selected to validate the proteomics results, but significant patient-to-patient variation obfuscated interpretation so corroborated the contradictory observations made by others. Conclusion Nevertheless, the study has provided significant insights into CC stage progression for further investigation.

Glioblastoma multiforme is the most aggressive malignant tumor of the central nervous system. Due to the absence of effective pharmacological and surgical treatments, the identification of early diagnostic and prognostic biomarkers is of key importance to improve the survival rate of patients and to develop new personalized treatments. On these bases, the aim of this review article is to summarize the current knowledge regarding the application of molecular biology and proteomics techniques for the identification of novel biomarkers through the analysis of different biological samples obtained from glioblastoma patients, including DNA, microRNAs, proteins, small molecules, circulating tumor cells, extracellular vesicles, etc. Both benefits and pitfalls of molecular biology and proteomics analyses are discussed, including the different mass spectrometry-based analytical techniques, highlighting how these investigation strategies are powerful tools to study the biology of glioblastoma, as well as to develop advanced methods for the management of this pathology.

Background: miRNAs (microRNAs) play a key role in triple-negative breast cancer (TNBC) progression, and its heterogeneity at the expression, pathological and clinical levels. Stratification of breast cancer subtypes on the basis of genomics and transcriptomics profiling, along with the known biomarkers&rsquo, receptor status, has revealed the existence of subgroups known to have diverse clinical outcomes. Recently, several studies have analysed expression profiles of matched mRNA and miRNA to investigate the underlying heterogeneity of TNBC and the potential role of miRNA as a biomarker within cancers. However, the miRNA-mRNA regulatory network within TNBC has yet to be understood. Results and Findings: We performed model-based integrated analysis of miRNA and mRNA expression profiles on breast cancer, primarily focusing on triple-negative, to identify subtype-specific signatures involved in oncogenic pathways and their potential role in patient survival outcome. Using univariate and multivariate Cox analysis, we identified 25 unique miRNAs associated with the prognosis of overall survival (OS) and distant metastases-free survival (DMFS) with &ldquo, risky&rdquo, and &ldquo, protective&rdquo, outcomes. The association of these prognostic miRNAs with subtype-specific mRNA genes was established to investigate their potential regulatory role in the canonical pathways using anti-correlation analysis. The analysis showed that miRNAs contribute to the positive regulation of known breast cancer driver genes as well as the activation of respective oncogenic pathway during disease formation. Further analysis on the &ldquo, risk associated&rdquo, miRNAs group revealed significant regulation of critical pathways such as cell growth, voltage-gated ion channel function, ion transport and cell-to-cell signalling. Conclusion: The study findings provide new insights into the potential role of miRNAs in TNBC disease progression through the activation of key oncogenic pathways. The results showed previously unreported subtype-specific prognostic miRNAs associated with clinical outcome that may be used for further clinical evaluation.

There 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 cancer-related 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 “omics” 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.

Rationale 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a low molecular weight drug of the flavonoid group, which has an anti-vascular effect in tumours causing endothelial cell apoptosis and activation of cytokines. Flavonoid-based compounds have been reported to lead to an upregulation in the expression of lysophosphatidylcholines (LPC)-type lipids in solid tumours. A study employing TLC/MALDI-MS and MALDI-MS imaging to examine LS174T colorectal adenocarcinoma xenografts following administration of DMXAA has been conducted into this effect. Methods LS174T colorectal adenocarcinoma xenografts grown in male immune-deficient mice were treated with 27.5 mg/kg DMXAA. The control (before treatment) and 4 h and 24 h post-treatment tumours were excised and divided into two. MALDI-MS imaging experiments were carried out on 12 µm cryosections sections taken from one half of the tumours and from the other half the lipids were extracted and analysed by TLC/MALDI-MS. These experiments were carried out in triplicate. Results Statistical analysis of the MALDI-MS imaging data set indicated an increased amount of LPC in the 24 h post-treated sample and a decreased amount of PC in the 24 h post-treated sample, compared with the 4 h post-treated sample and the control. These effects were confirmed by the TLC/MALDI-MS data. The lipid extracts were separated into six spots on the TLC plate. These were identified as arising from different lipids classes, i.e. LPC, sphingomyelins (SM), phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The TLC/MALDI-MS data indicated that LPC were highly expressed in the 4 h and 24 h post-treated tumour samples compared with the control. Examination of the mass spectrometric images confirms this increase and demonstrates additionally that the increase in the signals arising from LPC appears to be localised primarily within the central areas of the xenograft. Conclusions An increase in expression of LPC lipids in solid tumours treated with DMXAA has been demonstrated and shown to be localised in the central area of the tumour. Copyright © 2015 John Wiley & Sons, Ltd.

Purpose Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopatholigical features. Our aim, in this pilot study, was to investigate the intra- and inter-patient protein composition of nipple secretions and the implications for their use as liquid biopsies. Experimental design Matched pairs of NAF (n = 15) were characterised for physicochemical properties and SDS PAGE. Four pairs were selected for semi-quantitative proteomic profiling and trypsin-digested peptides analysed using 2D LC Orbitrap Fusion mass spectrometry. The resulting data was subject to bioinformatics analysis and statistical evaluation for functional significance. Results A total of 1990 unique proteins were identified many of which are established cancer associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals was observed. Conclusions This was the most complete proteomic study of NAF to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. This article is protected by copyright. All rights reserved

Aims: A sub-population of cells named cancer stem cells (CSCs) that initiate and promote tumour growth have been demonstrated to exist in several malignancies including colon carcinoma. The objective of our pilot study was to isolate CD133+CD26+CD44+ CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. Parallel cultures of non-cancer controls from colon normal lining and nonadenomatous polyps were set up. Methods: Magnetic activated cell sorting was used to isolate CD133+CD26+CD44+ cell populations followed by primary cell culturing under stem cell culture conditions. Number, cells/organoid and daughter generations of organoids were calculated using phase contrast microscope. Trypan blue exclusion method was used to test the viability of the cells. Results: Both colon tumour and colon non-adenomatous polyp formed floating organoids in suspension; however non-adenomatous polyp cultures did not show self-renewal properties for more than 1 passage. Normal colon singlecell suspension did not create organoids. Metastatic colon tumours rapidly produce cancer cell organoids in less than 24 hours in larger numbers compared to non-metastatic colon tumours (1-3 weeks). Metastatic colon tumour organoids have the ability for proliferation for upto five daughter generations in primary culture compared to three generations for those grown from non-metastatic tumours. Conclusions: This in vitro CSC organoid model will help study colon cancer biology, in particular providing a valuable source of primary cell-derived tissue for studying personalized molecular profiling using ‘omics strategies to direct therapeutic intervention.

Introduction: Cytochromes P450s (CYPs) constitute a superfamily of xenobiotic metabolising enzymes responsible for metabolism of many pharmaceuticals in the liver. Elevated mRNA levels of specific isoforms, such as CYP2S1, are associated with poor prognosis in colorectal cancer (CRC), and represent novel therapeutic targets for biotransformation of prodrugs to potent cytotoxics at the cancer site. In order to understand the expression of CYP2S1 protein, we have developed a parallel reaction monitoring mass spectrometry (PRM MS) assay to screen CRC samples. Method: Peptides (n=3) uniquely associated with CYP2S1 were synthesized and used as standards to optimise analytical performance (fragmentation conditions, LC retention time, LOQ, LOD, dynamic range) in an Orbitrap Fusion-based PRM MS assay. Levels of CYP2S1 were then determined in protein extracts of CRC, relative to the standards. Results: The PRM MS assay yielded quantitative data over 3 orders of magnitude. CYP2S1 was detected in C106, CaCO2, HCC2998, HT55 and DLD1 cell lines (0.05-1.08pg) and HT55 and DLD1 xenografts (0.29-0.41pg). CYP2S1 was also detected in patient-specific CRC tissues, with elevated levels in Stage III tumours. Conclusions: The PRM MS assay provides a specific, sensitive, high throughput method for identifying CYP2S1 compared to established enzyme and immunoassays. CYP2S1 levels vary considerably across CRC sources highlighting the importance of screening to identify the correct models for new drug development and the right patients for subsequent treatment. Citation Format: Sadr ul Shaheed, Laurence H. Patterson, Klaus Pors, Chris W. Sutton. Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4545.

Matched 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.

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.

The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.

Triple negative breast cancers (TNBC) are clinically heterogeneous, an aggressive subtype with poor diagnosis and strong resistance to therapy. There is a need to identify novel robust biomarkers with high specificity for early detection and therapeutic intervention. Microarray gene expression-based studies have offered significant advances in molecular classification and identification of diagnostic/prognostic signatures, however sample scarcity and cohort heterogeneity remains area of concern. In this study, we performed integrated analysis on independent microarray big data studies and identified a robust 880-gene signature for TNBC diagnosis. We further identified 16-gene (OGN, ESR1, GPC3, LHFP, AGR3, LPAR1, LRRC17, TCEAL1, CIRBP, NTN4, TUBA1C, TMSB10, RPL27, RPS3A, RPS18, and NOSTRIN) that are associated to TNBC tissues. The 880-gene signature achieved excellent classification accuracy ratio on each independent expression data sets with overall average of 99.06%, is an indication of its diagnostic power. Gene ontology enrichment analysis of 880-gene signature shows that cell-cycle pathways/processes are important clinical targets for triple negative breast cancer. Further verification of 880-gene signature could provide additive knowledge for better understanding and future direction of triple negative breast cancer research.

Recently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s12935-014-0143-3) contains supplementary material, which is available to authorized users.

Triple-negative breast cancers (TNBC) are clinically heterogeneous, an aggressive form of breast cancer with poor diagnosis and highly therapeutic resistant. It is urgently needed for identifying novel biomarkers with increased sensitivity and specificity for early detection and personalised therapeutic intervention. Microarray profiling offered significant advances in molecular classification but sample scarcity and cohort heterogeneity remains challenging areas. Here, we investigated diagnostics signatures derived from human triple-negative tissue. We applied REMARK criteria for the selection of relevant studies and compared the signatures gene lists directly as well as assessed their classification performance in predicting diagnosis using leave-one-out cross-validation. The cross-validation results shows excellent classification accuracy ratios using all data sets. A subset signature (17-gene) extracted from the convergence of eligible signatures have also achieved excellent classification accuracy of 89.37% across all data sets. We also applied gene ontology functional enrichment analysis to extract potentially biological process, pathways and network involved in TNBC disease progression. Through functional analysis, we recognized that these independent signatures have displayed commonalities in functional pathways of cell signaling, which play important role in the development and progression of TNBC. We have also identified five unique TNBC pathways genes (SYNCRIP, NFIB, RGS4, UGCG, LOX and NNMT), which could be important for therapeutic interventions as indicated by their close association with known drivers of TNBC and previously published experimental studies.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AA-reactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder.

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but

Introduction. Detection of the very earliest stages of breast cancer remain challenging and the differentiation of those carcinomas that develop into aggressive invasive forms from those that remain benign has not been achieved. Consequently, removal of the tumor by total mastectomy, local excision (LE) plus adjuvant radiotherapy (RT), or LE alone remain the main treatments, even if they prove unnecessary. Previously, using proteomics analysis, we identified proteins that were significantly changed in expression between matched normal and tumor tissues from Cypriot patients with different stages of the disease. Heat shock protein 10 (HSP10), which is responsible for the mitochondrial protein folding, was one of the proteins identified. A range of methods were used to verify HSP10 as a possible biomarker for breast cancer. Materials and Methods. Protein extracts from breast cancer cell lines and breast tissue biopsies from Cypriot patients with different stages of the disease were analysed by (i) Western Blotting (WB) and (ii) multiple reaction monitoring mass spectrometry (MRM MS) using two proteotypic peptides unique to HSP10, in proteins extracted form fresh frozen as well as from FFPE breast tissues. Tissue microarray analysis (TMA) was carried out on core biopsies from the Leeds Breast Tissue Bank. Results. WB indicated that HSP10 was ubiquitously and evenly expressed across breast cancer cell lines representative of different phenotypes. WB and MRM MS revealed that expression was significantly increased in invasive carcinoma compared to normal tissue, but was patient specific for fibroadenoma and DCIS cases. TMA of 360 samples indicated a strong correlation between staining and type of breast cancer (invasive ductal and lobular), grade and ER positive phenotype. Discussion. Proteomics analysis using iTRAQ quantitation indicated an average of 1.6 fold increase in HSP10 in invasive carcinomas compared to matched normal tissue. By comparison, WB and MRM MS analysis indicated a significantly amplified discrimination in the expression of HSP10 as has been observed with other biomarker candidates. HSP10 protein levels were in agreement with mRNA results observed in breast cancer cell lines reported in the Cancer Cell Line Encyclopaedia. In conclusion, HSP10 represents an excellent biomarker candidate for further validation. Citation Format: Sadr-ul Shaheed, Andreas Hadjisavvas, Kleitos Sokratous, Paul Loadman, Chris Sutton, Kyriacos Kyriacou. Analysis of HSP10 as a putative biomarker of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2015-1576

Introduction. Despite oestrogen and human epidermal growth factor receptor (HER2) targeted therapies improving patient survival, breast cancer continues to be a significant cause of premature death since many tumors do not respond to treatment or acquire resistance. Hence there remains the need to identify novel targets for therapy and biomarkers for prediction of response to treatment. A quantitative proteomics study of matched normal and tumor biopsies identified 63 proteins to be significantly increased or decreased in stage-specific tumors. Some have previously been associated with breast cancer, while others such as cofilin-1 represent new targets for investigation. Cofilin-1 was subject to a range of analyses to determine its association with breast cancer. Materials and Methods. Western blotting (WB) was performed on protein extracts from breast cancer cell lines and matched normal and tumor biopsies from Cypriot patients with different stages of the disease. Immunohistochemistry (IHC) was carried out on core biopsies from the Leeds Breast Tissue Bank. Multiple reaction monitoring (MRM) mass spectrometry was performed on trypsin-digested protein extracts from biopsies. Data from protein (Human Protein Atlas) and genomics (BioGPS, TiGER, UniGene) databases were assimilated with the experimental results. Results. WB indicated that cofilin-1 was ubiquitously expressed in tumor cell lines, representative of Luminal A, Luminal B, basal-like, claudin-low and HER2 phenotypes, at higher levels than normal breast cell lines. WB also indicated increased expression of cofilin-1 in invasive carcinoma tissues, compared to matched normal. Patients with ductal carcinoma in situ or fibroadenoma exhibited less clear results, either increasing slightly or remaining unchanged. MRM analysis of three cofilin-1 peptides in tissue extracts of invasive carcinoma patients indicated expression only in tumor. IHC of core biopsies exhibited strong staining for cofilin-1 in ductal invasive carcinoma tissues with no staining in normal breast cells. Genomics and proteomics databases indicate that cofilin-1 is expressed in a diverse range of normal tissues including major organs, gastro-intestinal tract, skin and tonsils. However, mRNA and protein levels are observed to be increased in skin, lung, liver, pancreatic and breast tumors. Discussion. The original quantitative proteomics data indicated only relatively small changes in expression (less than 2-fold) due to dynamic range limitations of the technique. Additional analytical approaches substantiated that cofilin-1 is significantly up-regulated in advanced breast cancer patients. Further patient sets are required to confirm the importance of cofilin-1 levels in early stages of breast cancer compared to benign tissues. Bioinformatics provided further confidence in our findings, highlighting the value of utilising databases for evidence of disease-specific proteins. Citation Format: Chris Sutton, Sadr ul-Shaheed, Paul Loadman, Valerie Speirs, Andrew Hanby, Andreas Hadjisavvas, Kyriacos Kyriacou. Expression profiling of cofilin-1 in breast cancer cell lines and biopsies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2013-2505

Introduction – Identification of markers of different stages of breast cancer remains a challenge. Although biofluids such as urine and plasma are preferred sources to discover potential biomarkers, tissue proteins are diluted compared to their site of expression and difficult to analyse due to degradation or due to the wide dynamic range of plasma proteins. As mastectomy is frequently used as part of treatment, it is possible to get sufficient histologically-defined tissue to perform proteomics profiling at the source of the disease and therefore more accurately identify changes in expression, for subsequent biomarker verification. Two dimensional separation of stable isotope-labelled total peptide mixtures from matched normal and disease biopsies from 10 patients with adenoma, DCIS or invasive carcinoma were compared in order to identify markers of different stages of the disease. Methods – Two lysis buffers, RIPA and urea, in combination with sonication, were used sequentially to maximise protein extraction from matched normal and diseased breast biopsies of 10 patients. The biopsies were split into three groups each comprising matched normal and diseased protein extracts from 4 patients one of which is common to all the groups to act as an internal control. Each protein extract in each group was trypsin-digested and iTRAQ (isobaric tag for relative and absolute quantitation) labelled. The digests from each group were pooled together and the resulting peptide mixture separated using isoelectric focusing followed by reverse phase nanoHPLC. Fractions from nanoHPLC were analysed by MALDI (matrix assisted laser desorption/ionisation) MS and MS/MS, and the data searched against a sequence database to identify proteins in the original sample. Results – The proteins identified in each group of 4 patients were compared and those which were observed in all three data sets, were subjected to further quantitative analysis. For each protein from each patient, a normal:tumor ratio was determined from the iTRAQ data. Those proteins for each patient that were significantly changed in expression (a ratio of 1.3 equates with a significant increase) were clustered together for further processing. Those proteins whose response was changed across a patient group (adenoma, DCIS or invasive carcinoma) were investigated for associations with cancer biology. Those which may represent novel disease stage-specific biomarkers were verified using immunohistochemistry and Western blotting. Discussion – A number of proteins significantly increased or decreased have previously been associated with breast cancer. In addition, there were a number of new proteins identified which were associated with adenoma, DCIS and invasive carcinoma stages of disease, and these may be suitable targets for drug development as well as for developing potential biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2011-4874

Hypoxia is a well established target for anticancer therapy. Within the tumor microenvironment, hypoxia does not reside alone; instead it exists in combination with multiple other pathophysiological features. The occurrence of these features in combination, presents a situation which is unique to tumors, and thus represents an attractive target for drug development. MCTS's provide an in vitro model system which encapsulates many of these features and enables the exploration of the molecular and biochemical changes mimicking those in vivo. Using a quantitative proteomics approach to characterise specific regions of the MCTS, it is hoped that novel targets for therapeutic interventions will be revealed. HT29 (colon adenocarcinoma) cell's were cultured as MCTS using spinner flasks. Spheroids were separated into 3 main fractions; the outer aerobic fraction (AF), the hypoxic fraction (HF) and the necrotic core (NC). This was achieved using serial trypsin treatments and gentle homogenisation. Proteins from each fraction were extracted and equal amounts from each were digested with trypsin. Resulting peptides were iTRAQ labelled and the fractions combined. The peptides were firstly initially separated according to their pI, using the Agilent OffGel system and then further separated by nano-HPLC, with fractions collected off-line onto a MALDI plate. MALDI TOF-TOF mass spectrometry was used to screen fractions in MS mode and a list of signals compiled for MS/MS analysis. The resulting MS/MS data was searched against protein sequence databases for identification. Validation of the proteomics results was achieved through western blot and Immunohistochemistry (IHC) analysis. The activity of a selection of proteins was also investigated using spectrophotometric assays. Confirmation of the spheroid separation methods was provided by hematoxylin and eosin staining and FAC analysis, where the necrotic core cells can be identified by their significantly smaller size. IHC using the endogenous hypoxia marker Carbonic Anhydrase IX was used to confirm the hypoxic status of these cells following their isolation. MS analysis resulted in 1031 protein identifications, of which 6.1 and 18.1% were shown at a higher level in the HF and NC, respectively, relative to the AF. Further, 4.9 and 9.8% of proteins were shown to be present at lower levels in the HF and NC, respectively, when compared to the AF. Activity assays, IHC and western blot analysis for selected proteins that significantly changed in expression showed good agreement with the MS-derived results. Many of those proteins identified as differentially expressed in the HF and NC represented established cancer associated proteins. Interestingly, a number of proteins, with no previous association with cancer, were shown to be up-regulated in both the HF and NC and may upon validation provide attractive leads for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4872. doi:10.1158/1538-7445.AM2011-4872

Introduction - Quantitative proteomic profiling of breast tissue has proven difficult due to its inherent histological complexity. Substantial differences in stroma, vasculature and cellular composition of normal and tumors have further exacerbated analysis. In order to address these issues, a novel strategy for proteomics analysis was developed to determine differences between normal and disease tissue and between patients with adenoma and invasive carcinoma. Methods - A dual lysis buffer method for extracting proteins, in combination with sonication, was used to maximise recovery from normal and diseased breast biopsies. Protein extracts from matched normal breast and tumors of three patients (one with adenoma and two with invasive carcinoma) were trypsin-digested, were iTRAQ (isobaric tag for relative and absolute quantitation) labelled, pooled and the resulting peptide mixture separated using isoelectricfocusing in the first dimension and reverse phase nanoHPLC in the second dimension. The eluate from nanoHPLC was collected onto matrix assisted laser desorption/ionisation plates, analysed by MALDI MS and MS/MS, the subsequent data searched against a sequence database to identify proteins in the original sample. Results - A total of 481 proteins with associated normal: tumor iTRAQ ratios for each patient. iTRAQ ratios were indicative of increased or decreased levels of each protein in tumor compared to normal tissue. Proteins were categorised by location as blood (13% of the identified proteins), extracellular (12%) and cellular (75%). The average ratios for each group of proteins provided a gross indication of the relative levels of each tissue component in the matched normal and tumor biopsies and demonstrated marked difference in adenoma compared to invasive carcinoma. iTRAQ ratios were normalised to enable comparison of significantly changed proteins between patients. There were many cellular proteins in common between the two invasive carcinoma patients (54 increased and 51 decreased in tumors) but few between either of the invasive carcinoma patients and the adenoma patient. Discussion - A number of proteins significantly increased or decreased have previously been associated with breast cancer (Tropomyosin beta chain, Periostin, Small breast epithelial mucin and Breast cancer estrogen-inducible protein were increased and Selenium-binding protein 1, Secreted frizzled-related protein 1 precursor and Kinectin were decreased), supporting the approach we have developed for proteomics analysis of breast tissues. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5555.

Introduction Hypoxia is a well established target for anticancer therapy. Within the tumor microenvironment, hypoxia does not reside alone; instead it exists in combination with multiple other physiological/biochemical changes. The occurrence of these features in combination, present a situation which is unique to tumors, thus represents an attractive target for drug development. MCTS's provide an in vitro model system which encapsulates many of these features. Using a quantitative proteomics approach to characterise specific regions of the MCTS, it is hoped that novel targets for therapeutic interventions will be revealed. Methods HT29 (colon adenocarcinoma) cell's were cultured as MCTS using spinner flasks. Spheroids were separated into 3 main fractions; the outer aerobic fraction (AF), the hypoxic fraction (HF) and the necrotic core (NC) by serial trypsinisation and gentle homogenisation. Proteins from each fraction were extracted and equal amounts from each fraction were digested with trypsin. Resulting peptides were iTRAQ labelled and the fractions combined. Peptides were firstly separated according to their pI, using the Agilent OffGel system and further separated by nano-HPLC. Fractions were collected off-line onto a MALDI plate. MALDI TOF-TOF mass spectrometry was used to screen fractions in MS mode and a list of signals compiled for MS/MS analysis. The resulting MS/MS data was searched against protein sequence databases for identification. Result Optimisation of the spheroid separation method revealed that 2 trypsin treatments were required to remove the AF of cells and 10-13 further trypsin treatments were required to reach the HF. Immunohistochemistry using the endogenous hypoxia marker Carbonic Anhydrase IX was used to confirm the hypoxic status of these cells following their isolation. Gentle homogenisation of the remaining spheroid shell, was shown to result in the optimal separation of the hypoxic cells from the non adherent cells present within the spheroid necrotic core. Confirmation of this separation was provided by hematoxylin and eosin staining, where the necrotic core cells can be identified by their significantly smaller size. MS analysis resulted in 882 protein identifications, of which 12.6% and 20.9% were shown at a higher level in the HF and NC, respectively, relative to the AF. Further, 8.4% and 8.7% of proteins were shown to be present at lower levels in the HF and NC, respectively, when compared to the AF. Conclusion Many of those proteins identified as differentially expressed in the HF and NC represented established cancer associated proteins. Interestingly, a number of proteins were highlighted as being up regulated in both the HF and NC which have no previous association with cancer and may upon validation, provide attractive leads for therapeutic intervention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4571.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups were discovered with extreme potency (IC50 16–640 nM) against the drug-resistant (Mitomycin C and EO9) colon carcinoma cell line BE, and also the human ovarian carcinoma A2780 and human non-small cell lung cancer H460 cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they are targeting the product of a ‘gene-like sequence’ of unidentified function. The identity of the target proteins was explored using an affinity chromatography proteomic strategy. Bespoke affinity matrices were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retain their activity (IC50 6.25 µM in a susceptible cell line, compared with > 100 µM for an insensitive cell line). The biotinylated ligands were exposed to protein extracts from the susceptible cell lines and ligand-protein complexes collected using a streptavidin-activated affinity column. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS/MS and identified using database searches. In parallel, the mechanism of action of the compound series has been investigated using NCI 3D MIND data mining. This suggests the mechanisms of action to be associated with phosphatase- and kinase-mediated cell cycle regulation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A204.

In situ investigation and characterisation of hypoxia-related protein markers in AQ4N treated tumour xenografts. MALDI-IMS-MSI enabled the visualisation of the distribution of AQ4N and AQ4 within the tissue sections, hence the selective localisation hypoxic tissue regions. Protein distribution and identification were obtained directly from AQ4N treated and non treated tumour tissue sections after in situ digestion.

BackgroundCurrently, there is an unmet clinical need in identifying and screening women at high risk of breast cancer, where tumours are often aggressive and treatment intervention is too late to prevent metastasis, recurrence and mortality. This has been brought into sharp focus by the SARS-CoV-2 global pandemic, constantly changing hospital policies and surgical guidelines in reducing access to established screening and treatment regimens. Nipple aspirate fluid (NAF), is thought to provide a unique window into the biological processes occurring within the breast, particularly in the context of a developing neoplasm. Evaluation of NAF in asymptomatic women, for novel chemical biomarkers of either early disease and/or cancer risk offers tremendous promise as a tool to facilitate early detection and to supplement screening. However, it is acceptability as a method of collection and screening by women is critical and yet unknown. A breast health questionnaire was disseminated to women through breast cancer charities, patient support groups and social media platforms, with the aim of collecting opinions on the acceptability of use of NAF as a potential screening tool.MethodFollowing ethical approval a questionnaire was prepared using online surveys consisting of four parts: (a) introduction on breast health screening in the UK, (b) core demographic data, (c) questions regarding screening and the acceptability of using NAF and (d) opinions about the process of collecting and using nipple fluid for screening. The voluntary and anonymous questionnaire was disseminated through social media, professional networks, charity websites and by individuals between October 2019 and December 2020. Survey responses were collected electronically, and the data analysed using online surveys statistical tools.ResultsA total of 3178 women completed the questionnaire (65.9% Caucasian, 27.7% Asian/British Asian, 0.6% black and 5.0% other). Of these, 2650 women (83.4%) had no prior knowledge of NAF and 89.4% were unaware that NAF can be expressed in up to 90% of all women. Concerning their risk of breast cancer, 89.8% of women were keen to know their future risk of breast cancer, 8.5% were unsure whether they wanted to know their risk and a further, 1.6% did not want to know. Regarding screening, 944 women (29.8%) were unaware of the lack of routine National Health Service Breast Screening for those under the age of 47 years. Furthermore, 53.0% of women were unaware that mammographic screening is affected by breast density. In terms of the acceptability of home testing for breast health, 92.0% were keen to undergo a home test. Both 79.7% and 70.9% stated they would consider hand massage and a breast pump to acquire nipple fluid samples, respectively. A further 48.6% of women would consider the use of a hormonal nasal spray for the same purpose. However, with regards to acquiring results from NAF testing, 42.6% of women would prefer to receive results at home and 34.2% in a medical facility. Finally, 91.6% of women believed that breast health should be incorporated as part of school education curriculum.ConclusionPublic awareness regarding breast screening protocols and limitations of mammography could be improved. Many women were unaware that NAF might be a useful biofluid for future risk prediction, and yet the concept of self-testing of nipple fluid, with either hand massage or a breast pump was well received. Efforts should be made to increase awareness of the benefits of alternative and supplementary tests, especially in the context of high-risk individuals and younger patients.