Personal Medicine and Bone Metastases: Biomarkers, Micro-RNAs and Bone Metastases.

Bone metastasis is a major cause of morbidity within solid tumours of the breast, prostate, lung and kidney. Metastasis to the skeleton is associated with a wide range of complications including bone fractures, spinal cord compression, hypercalcaemia and increased bone pain. Improved treatments for bone metastasis, such as the use of anti-bone resorptive bisphosphonate agents, within post-menopausal women have improved disease-free survival; however, these treatments are not without side effects. There is thus a need for biomarkers, which will predict the risk of developing the spread to bone within these cancers. The application of molecular profiling techniques, together with animal model systems and engineered cell-lines has enabled the identification of a series of potential bone-metastasis biomarker molecules predictive of bone metastasis risk. Some of these biomarker candidates have been validated within patient-derived samples providing a step towards clinical utility. Recent developments in multiplex biomarker quantification now enable the simultaneous measurement of up to 96 micro-RNA/protein molecules in a spatially defined manner with single-cell resolution, thus enabling the characterisation of the key molecules active at the sites of pre-metastatic niche formation as well as tumour-stroma signalling. These technologies have considerable potential to inform biomarker discovery. Additionally, a potential future extension of these discoveries could also be the identification of novel drug targets within cancer spread to bone. This chapter summarises recent findings in biomarker discovery within the key bone metastatic cancers (breast, prostate, lung and renal cell carcinoma). Tissue-based and circulating blood-based biomarkers are discussed from the fields of genomics, epigenetic regulation (micro-RNAs) and protein/cell-signalling together with a discussion of the potential future development of these markers towards clinical development.

Dedicator of Cytokinesis 4: A Potential Prognostic and Predictive Biomarker Within the Metastatic Spread of Breast Cancer to Bone.

Metastasis to bone occurs in over 70% of patients with advanced breast cancer resulting in skeletal complications, including pathological fractures, hypercalcaemia, and bone pain. Significant advances have been made in the treatment of bone metastases, including the use of antiresorptive drugs, such as bisphosphonates, as well as antibody-based therapies targeting key signalling intermediates within the process of cancer-mediated bone destruction. Despite these advances, treatment is not without side effects, including osteonecrosis of the jaw therefore biomarkers predictive of which patients are at high risk of developing bone spread are required to enable personalized medicine initiatives within this important disease area. We used proteomic analysis to compare the protein expression within (1) a parental triple negative human breast cancer cell line, (2) a fully bone homing cell line and (3) a lung homing cell line. The bone and lung homing cell-lines were derived by intra-cardiac injection of fluorescently labelled cells within immune-compromised mice. Proteomics identified Dedicator of Cytokinesis 4 as a biomarker predictive of bone spread, and this finding was further supported by the observation that high levels of Dedicator of Cytokinesis 4 within primary breast tumours were predictive of breast cancer spread to bone. Here, we provide an overview of this study and put the findings into context.

Identification and validation of DOCK4 as a potential biomarker for risk of bone metastasis development in patients with early breast cancer.

Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture-mass spectrometry; SILAC-MS) to compare protein expression in a bone-homing variant (BM1) of the human breast cancer cell line MDA-MB-231 with parental non-bone-homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC-MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone-homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4-shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06-4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176-3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4-expressing tumours. High DOCK4 expression was not associated with metastasis to non-skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CAPG and GIPC1: Breast Cancer Biomarkers for Bone Metastasis Development and Treatment.

BACKGROUND: Bone is the predominant site of metastasis from breast cancer, and recent trials have demonstrated that adjuvant bisphosphonate therapy can reduce bone metastasis development and improve survival. There is an unmet need for prognostic and predictive biomarkers so that therapy can be appropriately targeted. METHODS: Potential biomarkers for bone metastasis were identified using proteomic comparison of bone-metastatic, lung-metastatic, and nonmetastatic variants of human breast cancer MDA-MB-231 cells. Clinical validation was performed using immunohistochemical staining of tumor tissue microarrays from patients in a large randomized trial of adjuvant zoledronic acid (zoledronate) (AZURE-ISRCTN79831382). We used Cox proportional hazards regression, the Kaplan-Meier estimate of the survival function, and the log-rank test to investigate associations between protein expression, clinical variables, and time to distant recurrence events. All statistical tests were two-sided. RESULTS: Two novel biomarker candidates, macrophage-capping protein (CAPG) and PDZ domain-containing protein GIPC1 (GIPC1), were identified for clinical validation. Cox regression analysis of AZURE training and validation sets showed that control patients (no zoledronate) were more likely to develop first distant recurrence in bone (hazard ratio [HR] = 4.5, 95% confidence interval [CI] = 2.1 to 9.8, P < .001) and die (HR for overall survival = 1.8, 95% CI = 1.01 to 3.24, P = .045) if both proteins were highly expressed in the primary tumor. In patients with high expression of both proteins, zoledronate had a substantial effect, leading to 10-fold hazard ratio reduction (compared with control) for first distant recurrence in bone (P = .008). CONCLUSIONS: The composite biomarker, CAPG and GIPC1 in primary breast tumors, predicted disease outcomes and benefit from zoledronate and may facilitate patient selection for adjuvant bisphosphonate treatment.

Molecular histology of lung cancer: from targets to treatments.

Lung cancer is the leading cause of cancer-related death worldwide with a 5-year survival rate of less than 15%, despite significant advances in both diagnostic and therapeutic approaches. Combined genomic and transcriptomic sequencing studies have identified numerous genetic driver mutations that are responsible for the development of lung cancer. In addition, molecular profiling studies identify gene products and their mutations which predict tumour responses to targeted therapies such as protein tyrosine kinase inhibitors and also can offer explanation for drug resistance mechanisms. The profiling of circulating micro-RNAs has also provided an ability to discriminate patients in terms of prognosis/diagnosis and high-throughput DNA sequencing strategies are beginning to elucidate cell signalling pathway mutations associated with oncogenesis, including potential stem cell associated pathways, offering the promise that future therapies may target this sub-population, preventing disease relapse post treatment and improving patient survival. This review provides an assessment of molecular profiling within lung cancer concerning molecular mechanisms, treatment options and disease-progression. Current areas of development within lung cancer profiling are discussed (i.e. profiling of circulating tumour cells) and future challenges for lung cancer treatment addressed such as detection of micro-metastases and cancer stem cells.

The role of the tumor-microenvironment in lung cancer-metastasis and its relationship to potential therapeutic targets.

Non-small cell lung cancer (NSCLC) accounts for >80% of lung cancer cases and currently has an overall five-year survival rate of only 15%. Patients presenting with advanced stage NSCLC die within 18-months of diagnosis. Metastatic spread accounts for >70% of these deaths. Thus elucidation of the mechanistic basis of NSCLC-metastasis has potential to impact on patient quality of life and survival. Research on NSCLC metastasis has recently expanded to include non-cancer cell components of tumors-the stromal cellular compartment and extra-cellular matrix components comprising the tumor-microenvironment. Metastasis (from initial primary tumor growth through angiogenesis, intravasation, survival in the bloodstream, extravasation and metastatic growth) is an inefficient process and few released cancer cells complete the entire process. Micro-environmental interactions assist each of these steps and discovery of the mechanisms by which tumor cells co-operate with the micro-environment are uncovering key molecules providing either biomarkers or potential drug targets. The major sites of NSCLC metastasis are brain, bone, adrenal gland and the liver. The mechanistic basis of this tissue-tropism is beginning to be elucidated offering the potential to target stromal components of these tissues thus targeting therapy to the tissues affected. This review covers the principal steps involved in tumor metastasis. The role of cell-cell interactions, ECM remodeling and autocrine/paracrine signaling interactions between tumor cells and the surrounding stroma is discussed. The mechanistic basis of lung cancer metastasis to specific organs is also described. The signaling mechanisms outlined have potential to act as future drug targets minimizing lung cancer metastatic spread and morbidity.

Omic-profiling in breast cancer metastasis to bone: implications for mechanisms, biomarkers and treatment.

Despite well-recognised advances in breast cancer treatment, there remain substantial numbers of patients who develop metastatic disease, of which up to 70% involves spread to bone, resulting in skeletal complications which have a major negative impact on mortality and quality of life. Bisphosphonates and newer bone-targeted agents have reduced the prevalence of skeletal complications, yet there remains significant unmet clinical need, particularly for the development of more specific therapies for the prevention and treatment of metastatic bone disease, for the prediction of risk of its development in individual patients and for the prediction of response to treatments. Modern 'omic' strategies can potentially make a major contribution to meeting this need. Technological advances in the field of nucleic acid sequencing, mass spectrometry and metabolic profiling have driven progress in genomics, transcriptomics (functional genomics), proteomics and metabolomics. This review appraises the recent application of these approaches to studies of breast cancer metastasis (particularly to bone), with a focus on understanding how omic approaches may lead to new therapeutic options and to novel biomarker molecules or molecular signatures with potential value in clinical practise. The increasingly recognised need for rigorous sample quality control and both pre-clinical and clinical validation to meet the ultimate goals of clinical utility and patient benefit is discussed. Future directions of omic driven research in breast cancer metastasis are considered, in particular micro-RNAs and their role in the post-transcriptional regulation of gene function and the possible role of cancer-stem cells and epigenetic modifications in the development of distant metastases.

Serum aminoacylase-1 is a novel biomarker with potential prognostic utility for long-term outcome in patients with delayed graft function following renal transplantation.

Early identification and prognostic stratification of delayed graft function following renal transplantation has significant potential to improve outcome. Mass spectrometry analysis of serum samples, before and on day 2 post transplant from five patients with delayed graft function and five with an uncomplicated transplant, identified aminoacylase-1 (ACY-1) as a potential outcome biomarker. Following assay development, analysis of longitudinal samples from an initial validation cohort of 55 patients confirmed that the ACY-1 level on day 1 or 2 was a moderate predictor of delayed graft function, similar to serum creatinine, complementing the strongest predictor cystatin C. A further validation cohort of 194 patients confirmed this association with area under ROC curves (95% CI) for day 1 serum (138 patients) of 0.74 (0.67-0.85) for ACY-1, 0.9 (0.84-0.95) for cystatin C, and 0.93 (0.88-0.97) for both combined. Significant differences in serum ACY-1 levels were apparent between delayed, slow, and immediate graft function. Analysis of long-term follow-up for 54 patients with delayed graft function showed a highly significant association between day 1 or 3 serum ACY-1 and dialysis-free survival, mainly associated with the donor-brain-dead transplant type. Thus, proteomic analysis provides novel insights into the potential clinical utility of serum ACY-1 levels immediately post transplantation, enabling subdivision of patients with delayed graft function in terms of long-term outcome. Our study requires independent confirmation.

Proteomic studies of urinary biomarkers for prostate, bladder and kidney cancers.

Urine is an ideal body fluid for the detection of protein markers produced by urological cancers as it can be sampled noninvasively and contains secreted and directly shed proteins from the prostate, bladder and kidney. Major challenges of working with urine include high inter-individual and intra-individual variability, low protein concentration, the presence of salts and the dynamic range of protein expression. Despite these challenges, significant progress is being made using modern proteomic methods to identify and characterize protein-based markers for urological cancers. The development of robust, easy-to-use clinical tests based on novel biomarkers has the potential to impact upon diagnosis, prognosis and monitoring and could revolutionize the treatment and management of these cancers.

Skeletal metastasis in renal cell carcinoma: current and future management options.

Metastasis to the skeleton is common in advanced renal cancer and leads to debilitating skeletal complications including severe pain, increased fracture rate and spinal cord compression. The incidence of renal cell carcinoma is increasing by around 2% per year and recent advances in targeted anti-angiogenic therapy for advanced disease are expected to lead to longer survival times. The clinical management of metastatic bone disease in renal cell carcinoma therefore merits greater focus than hitherto. Bone metastases arising from renal cancer are highly osteolytic and particularly destructive. Fortunately, the continuing development of anti-resorptive drugs is revolutionising the medical management of metastatic bone disease across many tumour types and making a major impact on quality of life. The bisphosphonate zoledronic acid is now licensed for use in advanced renal cell carcinoma and appears to yield a greater benefit in terms of reduction in skeletal related events than in bone metastases arising from other tumour types. Drugs which are directed at specific targets in the bone metastasis pathway are in development, including denosumab, a fully human monoclonal antibody against receptor activator of nuclear factor kappa B ligand, which has recently been licensed in the United States for use in renal cell carcinoma, with European licensing expected soon. This review examines the increasing options for treatment of metastatic bone disease in renal cell carcinoma, with a focus on drug-based advances and progress in the development of existing and new biomarkers to support clinical management.

A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top-down and bottom-up proteomic analyses.

Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2-D DIGE ('top-down') and LC-MS/MS ('bottom-up'). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high-throughput 1-D-LC-MS/MS approach, including known biomarkers and moderate-lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2-D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC-MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label-free analysis, greater run-to-run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1-D nano-LC-LTQ Orbitrap Velos analysis in a moderate through-put biomarker discovery process.

CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

Application of proteomic analysis to the study of renal diseases.

Proteomics-based approaches are generating considerable data in clinical nephrology covering almost all aspects of the discipline. Proteomic experiments commonly involve fractionation and protein separation, followed by mass spectrometric analysis to identify proteins and peptides. Biostatistical and bioinformatical input is essential in such experiments, from initial experimental design to analysis of data. Standardization of procedures is an important research objective. Depending on study design, results can lead to biomarker discovery, mechanistic insight and identification of potential avenues for therapeutic intervention and treatment evaluation. Understanding proteomic information and its place in current clinical research and practice is fundamental. This Review describes proteomic experimentation and the concepts behind it, and gives an overview of its application to important areas in clinical nephrology including acute kidney injury, chronic kidney disease, end-stage renal disease, genetic diseases and fluid and electrolyte disorders, with a particular focus on biomarker discovery. The importance of future developments, such as the establishment of an infrastructure for a 'biomarker pipeline' with structured validation pathways for candidate biomarkers and development of clinical assays, is also discussed and some future perspectives are presented.