Recently, low-molecular-weight-peptide enrichment from blood samples by on-chip fractionation with nanopore platforms has been established successfully for the quantification and phenotypic characterization of the substrate degradome – the peptide products generated by the protease activity of a tumour environment. This article will provide evidence for this peptidomics-based approach and the clinical relevance in future therapeutic benefits will also be discussed.

by Dr Xu Qian and Dr Tony Y. Hu

Introduction
The development of cancer is a multistep process involving initiation, progression, local-regional recurrence, tumour metastasis and the host anti-tumour response. We are also now aware that changes in the broad genetic and epigenetic landscape as well as molecular mechanisms beyond histology and clinical characteristics contribute to this process. One such mechanism is the relationship between the repertoire of proteases expressed by a tissue and their substrates, which was found to be important in all steps of tumour progression by interactions with tumour cells and the tumour milieu.

Considering the systematic role of proteases in malignant tumour development, it was thought that it might be possible to detect signature products of substrate proteolysis – the substrate degradome – in the patient’s blood samples that are the result of protease dysregulation. This might then function as a diagnostic marker for tumour progression and a surrogate marker for monitoring the effects of protease-inhibitor therapy. This approach, called ‘exogenous peptidomics’ [1], based on mass spectrometry (MS) has proven its usefulness in the discovery of peptides from biofluids.

Challenges remain in this field as a consequence of the low molecular weight, low concentrations and quick degradation of such peptides in the peripheral blood of cancer patients. We recently developed an MS-based on-chip fractionation method assisted by nanopore technology, which has the advantages of being simple, high-throughput, high-resolution, and non-invasive [2]. We successfully identified circulating carboxypeptidase N (CPN)-catalysed C3f-fragments in a breast cancer mouse model as well as in patients with breast cancer [3] and matrix metalloproteinases (MMP)-9-catalysed C3f-fragments in an ovarian cancer mouse model [4]. This review discusses the applications of this new approach for studying peptide profiling in relation to tumour-resident proteases as biomarkers and potential therapy target.

Proteases and the substrate degradome in cancer development
The developing tumour microenvironment is composed of proliferating tumour cells, blood vessels, infiltrating inflammatory cells, a variety of associated tissue cells and tumour stroma, as well as secreted cytokines, chemokines, growth factors and matrix-degrading proteases. Intracellular and extracellular proteases that can function as signalling molecules play an indispensable role in this neoplastic process by enhancing cell proliferation, survival, adhesion, migration, angiogenesis, senescence, autophagy, apoptosis and evasion of the immune system in the tumour microenvironment [5–7]. For example, intracellular granzyme B is a well-known protease facilitating the ability of NK cells and CD8+ T-cells to kill their targets in the tumour milieu [8]. Elevated levels of granzyme B were also found in pre-metastatic niches presenting a novel role for the activation of CD8+ T-cells in constraining myeloid cell activity through direct killing [9]. Interestingly, recent publications suggested that granzyme B has a double-edged function. Regulatory T (Treg)-cells derived from the tumour environment may induce NK and CD8+ T-cell death in a granzyme B- and perforin-dependent fashion [10, 11] or kill CD4+ effector T-cells via granzyme B in the presence of IL-2 [12]. These findings indicate that granzyme B is relevant for Treg-cell-mediated suppression of tumour clearance in vivo.

Extracellular matrix (ECM) degradation by proteolysis is critical for tumour invasion and metastasis. Many proteases such as matrix metalloproteinases (MMPs), cathepsin and the urokinase-type plasminogen activator system play roles in the degradation of ECM in tumour progression [13, 14]. Extracellular granzyme B released from migrating cytotoxic lymphocytes was found to participate in the remodelling of vascular basement membranes (BMs) by cleaving BM constituents and enabling chemokine-driven movement through BMs in vitro [15]. Recently, another granzyme family member, granzyme M, was reported to be an inducer of epithelial-mesenchymal transition (EMT) in cancers associated with STAT3 activation [16]. Cancer cells with EMT features were capable of changing their shape, polarity and motility in a malignant manner. In the same study, overexpression of granzyme M in cancer cells was found to promote chemoresistance. The EMT phenotype of cancer cells was also achieved by increased MMP-9 production and MMP-9-mediated degradation of E-cadherin, involving ERK1/2 pathways [17].

In elucidating the role of proteases in cancer development, it is also important to gain a better understanding of the substrate degradome, which consists of the terminal peptide products of the activities of the multistage proteases. We have, therefore, identified hundreds of substrates of granzyme B, affecting cell lysis, receptors, cytokines and growth factors, as well as extracellular-matrix-structural proteins and intracellular proteins involved in cell signalling and cycle regulation [15, 18]. Besides granzyme B, MMPs cleave an increasingly large set of substrates, such as elastin, fibronectin, laminin and collagen IV [14, 19, 20]. However, given the broad range of substrate function, the mechanism of protease temporal and spatial regulation remains largely unknown. The exact role of proteases and substrates in cancer biology both at tissue level and in circulation is still needs to be clearly defined.

A new tool for the detection of circulating tumour-associated peptides as biomarkers
The proteolytic products of a protease are a useful indicator of the protease concentration in serum. This is necessary, in part, because of the low concentration of protease itself in serum compared with high detection limits, the quick degradation of the protease, and the inaccuracy of detection methodology such as enzyme linked immunosorbent assay (ELISA) as a result of irreversible non-specificity. New protocols are currently being developed for the detection and validation of substrate/peptides via peptidomics [21, 22].

Our group developed a platform mainly including peptide on-chip fractionation followed by a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis [23]. Briefly, nanoporous silica (NPS) thin films with nanotextures are used to capture and preserve low-molecular-weight peptides from 5 μl serum or plasma samples, whereas high-molecular-weight proteins are excluded by a wash step (Fig. 1). By this on-chip fractionation, low-molecular-weight peptides are enriched and separated. The peptides appear as m/z peaks that could be detected by MALDI-TOF MS analysis. The main advantage of this platform is that it allows specific profiling of the peptidome with high-throughput, high-resolution and a simple loading step.

Using this unique platform, we tested one hypothesis that, in breast cancer, CPN activity and its proteolytic products could be detected in interstitial fluid and blood [3]. CPN together with its substrate/peptide product levels may vary during tumour initiation and progression, indicating different disease states. We confirmed by ex vivo peptide cleavage assay and in vivo validation that the previously identified substrate C3f_S₁₃₀₄-R₁₃₂₀ was cleaved by CPN specifically at the C-terminal arginine. Moreover, six fragments generated from C3f_S₁₃₀₄-R₁₃₂₀ cleavage by CPN increased significantly in mouse sera at 2 weeks after orthotopic implantation relative to normal controls. The most important finding, however, documented that the plasma levels of substrate/peptide products of CPN were apparently elevated in patients with early stage breast cancer relative to controls, but levels of CPN protein itself were unchanged. These observations indicate that there may be additional regulation of CPN at different stages of tumour development. It is likely that inhibition of CPN protease activity is included within this additional regulation. However, the presence and frequency of substrate/peptides of CPN in the early stage of breast cancer makes them potential biomarkers for early diagnosis.

Recently, we investigated MMP-9 activity with the aim of monitoring a novel therapeutic strategy. To do this we used a HeyA8-MDR-induced ovarian cancer mouse model, where HeyA8-MDR cells are a human drug-resistant ovarian cancer cell line [4]. This study provided two major observations: (1) C3f was cleaved by MMP-9 in the tumour microenvironment. Two fragments generated specifically by this proteolysis were released and were detectable in mouse serum. (2) Treatment with ephrin type-A receptor 2-siRNA-multistage vectors (MSV-EphA2) induced apoptosis of tumour cells and a down-regulation of MMP-9 in tumour tissue. Moreover, the decreased level of circulating C3f cleavage fragments correlated with MSV-EphA2 treatment. Therefore, this change could be tracked and used to monitor treatment efficiency in real-time by a simple on-chip blood test. Taken together, these data suggest that the effect of EphA2 treatment extends to the peripheral blood, well beyond the tumour microenvironment at the tissue level, and thus can be easily assessed.

We reasoned that, from these two experimental approaches, it might be possible to gain information about the dynamic processes of proteases and their substrate/peptide products in patients with cancer. Consequently, further research in this field combined with other investigations aimed at improving the management of patients with cancer by early diagnosis, accurate characterization of disease, focused, treatment efficiency, and prognosis is essential.

Conclusion
In summary, MS-based on-chip fractionation assisted by nanopore platforms has been shown to be a highly sensitive and practical tool for the quantification and characterization of the circulating degradome. The combination of cellular protease function as well as substrate/peptide analysis provides a biologically meaningful picture of a specific tumour-entity at the level of the single peptide. Analysis of the circulating pepidome will be complementary to the standard diagnostic or prognostic procedure, such as routine blood test and tissue biopsy, for patients with cancer. This approach also holds great promise as a tool for monitoring novel therapeutic targets. For further development of this technique, many predicted targets still await validation as direct protease substrates and clarification of biological relevance in the network of protease and its inhibitors. We should pay much attention to the potential pitfalls.

References
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2. Fan J, Niu S, Dong A, Shi J, Wu HJ, Fine DH, et al. Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids. Nanomedicine. 2014; 10(5): 879–888.
3. Li Y, Li Y, Chen T, Kuklina AS, Bernard P, Esteva FJ, et al. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. Clin Chem. 2014; 60(1): 233–242.
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9. Zhang W, Zhang C, Li W, Deng J, Herrmann A, Priceman SJ, et al. CD8+ T-cell immunosurveillance constrains lymphoid premetastatic myeloid cell accumulation. Eur J Immunol. 2015; 45(1): 71–81.
10. Gondek DC, Lu LF, Quezada SA, Sakaguchi S, Noelle RJ. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J Immunol. 2005; 174(4): 1783–1786.
11. Cao X, Cai SF, Fehniger TA, Song J, Collins LI, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 2007; 27(4): 635–646.
12. Strauss L, Bergmann C, Whiteside TL. Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis. J Immunol. 2009; 182(3): 1469–1480.
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15. Prakash MD, Munoz MA, Jain R, Tong PL, Koskinen A, Regner M, et al. Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling. Immunity 2014; 41(6): 960–972.
16. Wang H, Sun Q, Wu Y, Wang L, Zhou C, Ma W, et al. Granzyme M expressed by tumor cells promotes chemoresistance and EMT in vitro and metastasis in vivo associated with STAT3 activation. Oncotarget. 2015; 6(8): 5818–5831.
17. Zuo JH, Zhu W, Li MY, Li XH, Yi H, Zeng GQ, et al. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin. J Cell Biochem. 2011; 112(9): 2508–2517.
18. Boivin WA, Cooper DM, Hiebert PR, Granville DJ. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab Invest. 2009; 89(11): 1195–1220.
19. Martinez A, Oh HR, Unsworth EJ, Bregonzio C, Saavedra JM, Stetler-Stevenson WG, et al. Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem J. 2004; 383(Pt. 3): 413–418.
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The authors
Xu Qian MD^1,2, Tony Y. Hu PhD*^1,3
1Dept of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA
²Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical University, Zhejiang, PR China
³Dept of Cell and Developmental Biology, Weill Cornell Medical College of Cornell University, NY 10065, USA

*Corresponding author
E-mail: yhu@houstonmethodist.org

Because of the critical nature of the reported clinical events (such as stent thrombosis and hemorrhage) associated with patients with certain cytochrome p450 2C19 (CYP2C19) variants receiving clopidogrel therapy, additional clinical studies are warranted. Large scale clinical trials seeking to correlate patient’s response to clopidogrel (Plavix®) with the CYP2C19 genotype may benefit from the inclusion of the CYP2C19 genotyping results into therapeutic anti-platelet therapy decisions before percutaneous coronary intervention.

by Dr H. Han, K. Blakely and Dr S. Lewis

Clinical potential of pharmacogenetic testing
The clinical potential of pharmacogenetic testing has increasingly been shown to influence treatment effectiveness for a number of therapies including tyrosine kinase inhibitors (TKIs) such as imatinib directed at chronic myelogenous leukemia (CML), gefitinib and erlotinib targeting the epidermal growth factor receptor (EGFR) in lung and other cancers and clopidogrel, an anti-platelet therapy prescribed for patients receiving percutaneous coronary intervention (PCI) [1–4].

Using genetic information in a clinically beneficial fashion, that is providing evidence-based data to show that the genetic information provided to the health care team is clinically applicable to diagnosis, treatment and prognosis and fiscally responsible, is challenging. Despite numerous studies relating genetic variants to clinical effects, the integration of genetics into routine clinical practice is restricted [5]. Success has been demonstrated especially in the area of oncology and many patients receive cancer therapy guided by genetic testing. Some examples of cancers that have therapy that may be guided by molecular testing include breast, lung, colon and leukemia. Obstacles to integration of genetic testing into routine practice include reimbursement issues and accessibility. Of particular concern to the cardiology team is the past unavailability of rapid point-of-care genotype testing for cytochrome p450 2C19 (CYP2C19) variants during PCI. Molecular genetic testing is typically performed at reference laboratories and the results of the testing have not been available to cardiologists at the time of PCI.
Moving to a state of personalized medicine and genome-guided care requires a number of important steps:

• Initially, single nucleotide polymorphisms (SNPs) must be discovered in the normal population. For the CYP2C19 gene, this includes greater than 2075 of the referenced SNPs.
• Secondly, SNPs must be related to phenotyped subjects in functional studies. For CYP2C19, currently the three clinically relevant variants appear to be *2,*3 and *17.
• Findings must be replicated in clinical settings demonstrating the advantage of providing the CYP2C19 genotype to guide practice at actionable times.
• Additional environmental, clinical and other genetic considerations must be evaluated to move to the personalized medicine of CYP2C19 drugs. In the case of CYP2C19 metabolized medications, a number of drugs may be prescribed to patients receiving PCI, including clopidogrel, omeprazole and aspirin. Both clopidogrel and omeprazole have narrow therapeutic ratios [6–9].

Clopidogrel therapy and genetic testing
The CYP2C19 enzyme is responsible for the metabolism of approximately 15% of all prescription drugs including anti-platelet therapies such as clopidogrel, beta-blockers (propranolol), anti-depressants (imipramine), anti-convulsants (phenytoin) and proton pump inhibitors (omeperizol) [6]. Of particular interest to cardiologists is anti-platelet therapy. Anti-platelet therapy may be classified according to the target of action and includes ADP antagonists (clopidogrel, prasugrel and ticlopidin), COX inhibitors (aspirin), phosphodieterase inhibitors (dipyridamole) and GP IIb/IIIa inhibitors (tirofiban, eptifibatide, abcixmab) shown in Figure 1. Clopidogrel bisulfate is a thienopyridine irreversible inhibitor of ADP-induced platelet aggregation by directly preventing ADP binding to its receptor P2Y12 and thereby preventing subsequent activation of the of the glycoprotein IIb/IIIa complex (Fig. 2). Platelets are irreversibly inactive for the remainder of their life, approximately 7–10 days [6].

Metabolism of clopidogrel (Fig. 3) occurs in the liver by several p450 enzymes. CYP2C19 is involved in the formation of the primary inactive metabolite, 2-oxo-clopidogrel and the active thiol clopidogrel deritive. Anti-platelet effects may be seen 2 hours after oral administration with a steady state inhibition reached between days 3–7. Anti-platelet effects are dose dependent and may be measured by platelet aggregation assays and differ according to CYP2C19 genotype. This association of CYP2C19 genotype and clopidogrel treatment outcome was evaluated in several clinical trials [2, 10].

Variants of the CYP2C19 enzyme include both reduced drug metabolism variants (*2,*3,*4,*5) and increased drug metabolism variants (*17). Most patients undergoing the insertion of a drug eluting stent after myocardial infarction are prescribed clopidogrel bisulfate (Plavix ^®) and aspirin as anti-platelet therapy. Numerous investigations of patients prescribed the anti-platelet drug clopidogrel bisulfate (Plavix^®) have demonstrated relationships between the patient’s CYP2C19 genotype and their response to clopidogrel as related to clinical outcomes such as blood clots, stent thrombosis, bleeding, myocardial infarctions and major cardiovascular events (MACE). Loss of function CYP2C19*2 and *3 variants have been associated with higher levels of ADP-induced platelet aggregation in patients receiving clopidogrel therapy, and therefore have a greater risk of major cardiovascular events, including stent thrombosis. Several clinical variables are implicated in the platelet response to clopidogrel, but the strongest predictor is the loss of function CYP2C19*2 allele. Approximately 30% of western European individuals, and 50% of Asian individuals carry the *2 allele and studies have associated the *2 allele with a significant increased risk of adverse cardiovascular events and stent thrombosis [11, 12].

In March of 2010, the FDA announced a boxed warning to the clopidogrel label, alerting patients and clinicians that clopidogrel may be less effective in patients carrying the reduced function alleles [13].

The CYP2C19*17 gain-of-function allelic variant was shown to significantly reduce ADP-induced platelet aggregation in clopidogrel-treated patients and was therefore predicted to confer an increased risk of bleeding. CYP2C19*17 alleles are found in the United States population ranging from <5% homozygotes to ~40% heterozygotes. The most common CYP2C10*17 variant, -806C>T, is associated with recruitment of a transcription factor to the mutated site, enhancing transcription and expression of the CYP2C19 enzyme. [14]

Current and future options for CYP2C19 testing
Currently, molecular reference laboratories can provide CYP2C19 genotyping results in several days. Recently, the Spartan RX point-of-care instrument was FDA cleared for in vitro diagnostic testing. This platform uses buccal cells and can provide results in about one hour [15].

Because of the past inability of molecular laboratories to provide cardiologists with rapid point-of-care testing for CYP2C19 variants, large-scale clinical trials with rapid genetic results provided to clinicians at the time of the PCI have been limited. Currently, a clinical trial (NCT01742117) is sponsored by the Center for Individualized Medicine at Mayo Clinic and is entitled ‘Tailored Antiplatelet Initiation to Lessen Outcomes due to Clopidogrel Resistance after Percutaneous Coronary Intervention’ (TAILOR-PCI). This randomized prospective study will use the FDA-cleared Spartan RX to genotype CYP2C19 for the *2,*3 and*17 alleles in cardiac stent patients. The carriers will receive ticagrelor instead of clopidogrel. Approximately 6000 patients will be enrolled in this large study with a completion date of June 2016. Results of this study should provide additional risk stratification and treatment decisions. Additional trials to formulate strategies for the most effective and cost efficient anti-platelet treatment will hopefully follow [15].

Advances in next-generation sequencing (NGS) are providing the promise of available genetic information on patients at rapidly reduced costs. Clinical applications of NGS will hopefully include pharmacogenetic information about patients that may be accessed by clinicians through the electronic medical record to provide immediate guidance of optimum therapy for not only anti-platelet therapy but for numerous other medications.

References
1. Gurbel PA, Tantry US. Controversies in cardiovascular medicine. Platelet function testing and genotyping improve outcome in patients treated with antithrombotic agents. Circulation 2012; 125: 1276–1287.
2. Sibbing D, Koch W, Gebhard D, et al. Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogrel-treated patients with coronary stent placement. Circulation 2010; 121: 512–518.
3. Krishna V, Diamond GA, Kaiil S. Do platelet function testing and genotyping improve outcome in patients treated with antithrombotic agents?: the role of platelet reactivity and genotype testing in the prevention of atherothrombotic cardiovascular events remains unproven. Circulation 2012; 125: 1288–1303.
4. Mega JL, Close SL, Wiviott SD, et al. Cytochrome p-450 polymorphisms and response to clopidogrel. N Eng J Med. 2009; 360: 354–362.
5. Roberts JD, Wells GA, Le May ML, et al. Point-of-care genetic testing for personalization of antiplatelet treatment (RAPID GENE): a prospective, randomized, proof-of-concept trial. Lancet 2012; 379: 1705–1711.
6. Plavix package insert. Bristol-Myers Squibb 2009.
7. Tantry U, Kereiakes D, Gurbel P. Clopidogrel and proton pump inhibitors. influence of pharmacological interactions on clinical outcomes and mechanistic explanations. JACC Cardiovasc Interv. 2011; 4: 365–380.
8. Gurbel PA, Tantry US, Shuldiner AR. Letter by Gurbel et al. regarding article, “Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogrel-treated patients with coronary stent placement”. Circulation 2010; 122: e478.
9. Sibbing D, Koch W, Gebhard D, et al. Response to letter regarding article, “Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogrel-treated patients with coronary stent placement”. Circulation 2010; 122: e479.
10. Taubert D, Kastrati A, Harlfinger S, et al. Pharmacokinetics of clopidogrel after administration of a high loading dose. Thomb Haemost. 2004; 92: 311–316.
11. Simon T, Verstuyft C, Mary-Krause M, et al. Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med. 2009; 360:363–375.
12. Brilakis ES, Patel VG, Banerjee S. Medical management after coronary stent implantation: a review. JAMA 2013; 310: 189–198.
13. Holmes DR Jr, Dehmer GJ, Kaul S, et al. ACCF/AHA Clopidogrel clinical alert: approaches to the FDA “boxed warning”: a report of the American College of Cardiology Foundation Task Force on clinical expert consensus documents and the American Heart Association endorsed by the Society for Cardiovascular Angiography and Interventions and the Society of Thoracic Surgeons. J Am Coll Cardiol. 2010; 56: 321–341.
14. Rudberg I, Mohebi B, Hermann M, et al. Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients. Clin Pharmacol Ther. 2008; 83: 322–327.
15. Spartan Bioscience announces 6,000-patient study of personalized medicine for cardiac stents. PRWeb 2012. (http://www.prweb.com/releases/2012/12/prweb10252073.htm)

The authors
Heping Han MD, PhD, MB (ASCP);
Katherine Blakely BS; Sally Lewis* PhD, MLS (ASCP), MB
Tarleton State University, Fort Worth,
TX 76104, USA

*Corresponding author
E-mail: slewis@tarleton.edu

Effective screening strategies have not yet been developed for the early detection of ovarian cancer. The serum biomarker CA125, routinely used to aid diagnosis and monitor treatment response, is not informative in all patients. Recent analytical developments have prioritized promising candidate novel biomarkers or multi-biomarker panels for future clinical evaluation.

by E. L. Joseph, Dr M. J. Ferguson and Dr G. Smith

Introduction to ovarian cancer
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and the fifth leading cause of cancer related death among women with around 140 000 annual deaths worldwide. EOC can develop as one of four histotypes with the serous histotype being the most common and most aggressive. The remaining three non-serous histotypes, endometrioid, clear cell and mucinous cancers present less frequently. High grade serous ovarian cancer, heterogeneous in nature and rapidly progressive, has a poor prognosis, where a major contributing factor is the lack of ability to diagnose the disease at a sufficiently early stage to facilitate curative surgery. The 5-year survival rate is less than 30% for patients presenting with advanced disease spread beyond the ovaries (FIGO Stage 3/4), but if detected earlier combination therapy of cytoreductive surgery and adjuvant or neo-adjuvant chemotherapy with platinum and taxane-based drugs has the potential to cure 90% of patients [1]. Consequently the identification of biomarkers capable of detecting ovarian cancer at the earliest stages and monitoring disease progression are inherently important in tackling this lethal disease. Due to its prevalence, the ideal biomarker for detecting early stage ovarian cancer requires an extremely high specificity (>99%) and a minimum sensitivity of 75% [2]. Despite extensive research, no optimal ovarian cancer biomarker has yet been identified and such high specificity is unlikely to be met by a single agent. Many promising candidate biomarkers are however currently undergoing evaluation in clinical trials.

CA125
The only ovarian cancer biomarker routinely used in the clinic is cancer antigen 125 (CA125; mucin 16) currently considered the ‘gold standard’ cancer biomarker despite its limitations. In the majority of patients with EOC, expression of the CA125 glycoprotein is raised above the normal reference range (>35 U/ml blood), but it only has a sensitivity of 50% to 60% with a specificity of 90% in early stage postmenopausal patients [3]. Several factors, however, limit the utility of CA125 in routine population screening: it is not expressed in 20% of ovarian cancers, is only significantly elevated in 47% of early stage ovarian cancers (although increasing to 80–90% in advanced stage cancers) and can be raised in many benign conditions including endometriosis and peritonitis. Variability in CA125 expression throughout the menstrual cycle and in pregnancy is also a common confounding issue. A major clinical utility of CA125, however, is related to its ability to commonly reflect clinical response following chemotherapy treatment and as such is often successfully used to monitor a patient’s progress through chemotherapy. A reduction in CA125 expression during treatment is considered a positive prognostic outcome for the patient and serial serum measurements are currently used to predict therapeutic outcomes and estimate stability of the disease (Fig. 1).

Biomarkers under evaluation
There have now been multiple attempts to identify novel ovarian cancer biomarkers with varying success. The most promising serum biomarkers include HE4 and mesothelin.

HE4
Human epididymis protein 4 (HE4) has been shown to be consistently elevated above the normal level (151 pM) in ovarian cancers, with sensitivity of 95% and specificity of 73%. HE4 is differentially expressed in specific subtypes of ovarian cancer, potentially allowing clinicians to distinguish histotypes to aid treatment; HE4 was found to be overexpressed in 100% of endometrioid cancers, 93% of serous cancers but only 50% of clear cell cancers [4]. Unlike CA125, it is less likely to produce false positives in benign masses and it has also been proposed to be the best candidate biomarker for early detection of Stage I disease despite sensitivity and specificity of 46% and 95% respectively [2]. HE4 has recently obtained FDA approval in the USA for monitoring recurrence or progression of EOC and, in comparative tests, has been found to be superior to CA125 in classifying benign and borderline ovarian cancers.

Mesothelin
Mesothelin is a glycoprotein expressed by mesothelial cells, the expression of which has been found to be raised in mesothelioma, pancreatic and ovarian cancers.

It can be easily measured in both urine and serum, highlighting its potential as a non-invasive biomarker. Serum mesothelin levels were found to be increased in approximately 60% of ovarian cancers with 98% specificity.

One study found elevation of mesothelin in 42% of urine assays as opposed to 12% serum assays of early stage EOCs at 95% specificity which reinforces the potential of this glycoprotein as an early detection biomarker and the use of urine in preference to serum [2]. Higher levels of mesothelin were also found to be associated with poorer overall survival in patients following optimal debulking surgery or who have advanced stage ovarian cancer. A recent study, however, revealed that lifestyle choices such as smoking and BMI can affect mesothelin levels, which also often increase with age.

Identification of new candidate biomarkers
Due to an urgent need for better biomarkers for early detection of ovarian cancer and reliable biomarkers to monitor clinical response, ongoing efforts are focused on the application of state of the art technologies e.g. mass spectrometry and quantitative proteomic analysis to identify novel biomarkers [5]. These approaches however often generate multiple candidate biomarkers for further investigation, prioritization and clinical evaluation of which is an ongoing challenge. These methods allow comparison of multiplex biomarker panels and identification of novel differentially expressed proteins not previously linked to ovarian cancer.

Another powerful technology is microarray-based mRNA analysis which allows genome wide expression studies which have already enhanced the understanding of the genes and pathways which influence ovarian cancer progression, chemotherapy response and survival. For example, the candidate biomarkers osteopontin and kallikrein (Table 1) were discovered by this method.

Our own studies have revealed significant differences in the expression of fibroblast growth factor 1 (FGF1) and additional FGF pathway genes in ovarian cancers of different histologies (Fig. 2A) and in paired sensitive and resistant ovarian cancer cell lines (Fig. 2B). We have additionally shown that FGF1 expression is significantly inversely correlated with both progression-free (Fig. 2C) and overall survival in ovarian cancer patients [6]. We are therefore currently recruiting patients to longitudinal clinical studies to investigate whether FGF1 or additional related growth factors can predict disease progression and/or the development of treatment-limiting drug resistance.

MicroRNAs (miRNAs) are small non-coding RNAs (19–25 nucleotides) that regulate gene expression by binding to mRNA target sequences and disrupting translation [7]. MiRNAs have great potential as diagnostic and clinical response biomarkers in ovarian and additional cancers as miRNA expression can now routinely be quantitatively assessed in small biopsies and in formalin-fixed material. For example, approximately 30 miRNAs (including miR-21, miR-141, miR-203, miR-205 and miR-214) are differentially expressed in ovarian cancer [8], while miRNAs including miR-200a, miR-200b and miR-429 have also been associated with cancer recurrence and have been shown to predict survival. For example, high expression of miR-200, miR-141, miR-18a and low expression of let-7b, and miR-199a were found to predict poor survival in a cohort of 20 ovarian cancer patients [9]. Meanwhile, recent data from our own laboratory has identified multiple miRNAs including miR-125b and miR-130 associated with the development of platinum resistance. MiRNAs are particularly promising candidate biomarkers due to their stability, and abundant expression in solid cancers, whole blood and routinely collected plasma and serum samples.

Future directions
Due to the challenges of finding a single biomarker that can encompass the complexity and heterogeneity of ovarian cancer it is logical that optimization of a multi-biomarker panel may be the most practical approach, for example combining HE4 and mesothelin with CA125 to augment both sensitivity and specificity. This type of approach has recently been proposed in algorithms such as the Risk of Ovarian Malignancy Algorithm or ROMA which combines CA125 and HE4 levels with a sensitivity of 94% and specificity of 75%. [4]. Combinations of CA125 and mesothelin have also been found to detect more cancers than each biomarker alone. Several current studies have, however, suggested that combination biomarker analysis significantly increases the predictive power of CA125, but also unfortunately appears to decrease specificity. Ongoing studies therefore aim to develop improved biomarker panels suitable both for early detection and treatment guidance of ovarian cancer (Table 2). All of these results still require validation but they are indicative of the possible power of using a multi-biomarker panel in diagnostic tests and for monitoring the clinical responses of ovarian cancer.

Concluding remarks
An ideal biomarker for ovarian cancer will have a high enough sensitivity to correctly diagnose women with the disease and be specific enough to avoid false positive results. With ongoing efforts to identify biomarkers which match this ideal, hundreds of candidates with clinical relevance have been found but still require much validation before having a routine place in the clinic. It is expected that the future of ovarian cancer detection will be based on panels of combination serum-based biomarkers alongside biological imaging techniques to improve diagnosis, treatment and disease management.

References
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14. Lokshin AE, Winans M, Landsittel D, Marrangoni AM, Velikokhatnaya L, Modugno F, Nolen BM, Gorelik E. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecol Oncol. 2006; 102: 244–251.

The authors
Emma L. Joseph¹ BSc; Michelle J. Ferguson² MBChB, MD; and Gillian Smith¹* PhD
¹Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee UK
²Tayside Cancer Centre, Ninewells Hospital & Medical School, Dundee UK

*Corresponding author
E-mail: g.smith@dundee.ac.uk

Recent findings indicate that aspects of high-density lipoprotein (HDL) not captured by traditionally measured HDL–cholesterol levels (HDL‑C) are likely to be cardioprotective. This review will highlight some of these studies and suggest new directions to identify the specific molecules that are responsible for the cardioprotective nature of HDL.

by Daniel S. Kim, Dr Patrick M. Hutchins and Prof. Gail P. Jarvik

Raising HDL-C does not confer cardioprotection
There is a well-established inverse association between high-density lipoprotein–cholesterol (HDL-C) levels and cardiovascular disease (CVD) in epidemiological and clinical studies [1, 2]. This robust relationship suggested that HDL-C was in the causal pathway of atheroprotection. Indeed a large number of studies have demonstrated that HDL possesses various anti-atherogenic properties, primarily the ability to accept cholesterol from macrophages in a process termed reverse cholesterol transport [3, 4].

In contrast, several high-profile studies have demonstrated that increasing levels of HDL-C does not have a significant cardioprotective effect. In a large and well-conducted clinical trial of the cholesterol ester transport protein (CETP) inhibitor, torcetrapib, there was no reduction in the incidence of CVD-related events despite significantly higher HDL-C levels [5]. A follow-up study using a different CETP inhibitor, dalcetrapib, also showed increased HDL-C levels yet there was no significant difference in CVD event rate between the treatment and placebo groups [6]. In a third randomized clinical trial that used niacin to increase HDL-C levels, there was again no reduction in cardiovascular events [7]. Finally, a large-scale Mendelian randomization study of approximately 20 000 myocardial infarction (MI) cases and 100 000 controls, showed that a genetic polymorphism which associated with approximately 10% higher HDL-C levels was not associated with decreased incidence of MI [8], again suggesting that the relationship between HDL-C and the prevention of cardiac events is not causal.

HDL particle concentration is a superior predictor of CVD
As the elevation of HDL-C was not beneficial in these studies, some have speculated that HDL itself is not cardioprotective. An alternative explanation for these negative data is that the cholesterol content of HDL – a surrogate measure of HDL – does not best reflect the anti-atherogenic properties of HDL. To resolve these issues it is critical to identify new HDL metrics that reliably reflect its cardioprotective functions.

One promising approach for assessing the role of HDL in CVD is to evaluate the individual HDL particles. HDL is a heterogeneous mixture of lipoprotein particles composed of discrete subspecies that have unique structural compositions and biological functions. As different HDL particles carry vastly different amounts of cholesterol – ranging over an order-of-magnitude [9, 10] – measuring the total HDL-C does not provide information regarding the distribution of HDL subpopulations or the number of total HDL particles.

HDL can be fractionated based on a number of physicochemical properties, most commonly size or density. Several techniques, both qualitative and quantitative, have been developed for HDL subspecies analysis. The various HDL subspecies reported by these techniques and their associated nomenclature are briefly summarized in Table 1 [see also ref. 11]. Furthermore, HDL subspecies determined by ultracentrifugation and calibrated ion mobility analysis (both are discussed in detail later) are shown in Figure 1. Many studies have demonstrated the potential clinical utility of HDL subspecies analysis, which can be achieved by techniques such as 2D gradient gel electrophoresis [12] and nuclear magnetic resonance (NMR) [13]. For example, one study (using 2D gradient gel electrophoresis) showed that very-large, cholesterol-rich α-1 HDLs were better predictors than HDL-C levels of reduced coronary heart disease (CHD) in a subset of males from the Framingham Offspring Study [14]. Another high-profile study, using NMR to assess HDL subspecies in over 2200 participants in the EPIC-Norfolk cohort, showed that higher HDL particle (HDL-P) concentrations were a predictor of reduced CHD, independent of classic CHD risk factors [15]. In more recent work from the Multi-Ethnic Study of Atherosclerosis, total HDL-P (measured by NMR) and HDL-C were evaluated at baseline for 5598 participants, who were then followed prospectively for incident CHD (n=227 events) [16]. Although both HDL-P and HDL-C were highly correlated with each other, in multivariate regression models total HDL-P concentration was the superior predictor of reduced incident CHD when compared to HDL-C. This finding indicates that although HDL-C captures a large portion of HDL-P variation, HDL-P is the better predictor of CHD.

These studies support the notion that measuring individual HDL particle subspecies provides clinically useful information beyond traditionally measured HDL-C. However, both α-1 HDLs (which are cholesterol-rich) and HDL-P measured by NMR (which relies on lipid to generate signal) are highly correlated with HDL-C. Therefore, it is possible that these observations reflect a similar inverse association observed between HDL-C and cardiovascular disease. Importantly, two recent studies (discussed below) indicate that low levels of relatively cholesterol-poor, smaller HDLs also associate with cerebrovascular disease, again suggesting that subspecies of HDL not adequately captured by measuring HDL-C may also play important roles in the pathogenesis of atherosclerotic disease.

Shifting focus: HDL-3 and medium-HDL particles
We investigated the association of the subspecies HDL-2 and HDL-3 (Table 1; Fig. 1) with carotid artery disease (CAAD) [17]. Here, HDL was sub-fractionated by ultracentrifugation and the subspecies were quantified by their cholesterol content. In a case-control cohort of 1,725 participants [part of the Carotid Lesion Epidemiology And Risk (CLEAR) cohort], stepwise linear regression was used to determine whether total HDL-C, HDL-2 cholesterol (HDL-2C), HDL-3 cholesterol (HDL-3C), or apolipoprotein A-I (apoA-I) levels were the best predictor of CAAD. In this study, the smaller HDL-3C fraction was found to be the best predictor of reduced CAAD risk. Moreover, adding HDL-3C to the model improved prediction even when HDL-C levels were also considered, demonstrating added utility of the HDL-3C measure versus HDL-C.

In a separate study using calibrated ion mobility analysis, the particle concentrations of three HDL subspecies (Table 1; Fig. 1) were measured in a subset of the same CLEAR cohort [18]. Participants with severe carotid stenosis (n=40; >80% stenosis by ultrasound in either or both internal carotid artery) had significantly lower plasma concentrations of medium-HDL particles compared with control participants (n=40; <15% stenosis by ultrasound in both carotid arteries). In this population HDL-P was a superior predictor of CAAD compared to HDL-C and this relationship was significant after controlling for HDL-C. The case-control difference in total HDL-P was driven by dramatic changes in medium-HDL particles, the next best predictor of CAAD. This medium-HDL particle inverse association also remained significant after controlling for HDL-C. Considering HDL-3 is composed of small- and medium-HDL particles (Fig. 1) and medium-HDL contributes the majority of HDL-3 cholesterol content, these results are in excellent agreement with the previous study of the CLEAR cohort. Both results support the hypothesis that relatively cholesterol-poor, smaller HDL subspecies, which are under-represented by total HDL-C, are potentially important protective factors for CVD.
Summary and future directions
Considering that increased levels of cholesterol-poor HDL subspecies – reflected by measures of HDL-3, medium size particles, and increased HDL-P – can represent superior predictors of CVD phenotypes, it is possible that pharmacologic attempts to raise HDL-C fail to affect CVD event rates because specifically elevating the cholesterol content of HDL is insufficient. The mechanism of HDL-C elevation should be considered. The agents tested thus far may have increased HDL-C by forming large, cholesterol-rich HDL particles at the expense of medium- and small-HDL particles; having an overall null effect on total particle concentration. Indeed, there is evidence from 2D-gel electrophoresis that very high HDL-C levels observed in CETP deficiency result from a shift from small- and medium-HDLs to large-HDL particles [19]. Thus, HDL directed therapies – especially CETP inhibitors – might increase HDL-C without increasing the number of total HDL-P and possibly reducing the number of potentially beneficial medium-HDL particles. Considering that medium- and total HDL particle concentrations may represent superior predictors of cardioprotection, this hypothesis could explain the failures of the CETP inhibitors and niacin to prevent CVD. We speculate that HDL directed therapies might be more effective in reducing CVD-related events if the number of circulating HDL particles was increased by therapy, especially medium-HDLs.

In light of recent research showing that certain subspecies of HDL (such as medium-HDL and HDL-3) may specifically contribute to cardioprotection, it is our opinion that the focus of research and potential therapies should shift to these promising targets. Of particular interest is the protein cargo of these HDL subspecies, which may reveal important mechanisms related to their cardioprotective properties. For instance, HDL-3 is closely associated with PON1 enzyme activity [20], which is associated with cardioprotection [21, 22]. Notably, the cardioprotective association of HDL-3 was in part independent of both PON1 activity and HDL-C, indicating that there were unmeasured predictive elements of the HDL-3 proteome; these may be apolipoproteins, or ancillary proteins that are specifically associated with HDL-3 [17].

In summary, it is our opinion that the recent failure of increased HDL-C to be cardioprotective likely reflects the fact that increasing HDL-C alone does not adequately increase the concentration or activity of cardioprotective HDL subspecies. It would be an error to say that studies of HDL-C demonstrate that HDL is not cardioprotective. Increased total HDL particle concentration, or perhaps a specific increase in medium-HDL particles, may confer greater protection against CAAD and CHD than pharmaceutically generating a preponderance of large, cholesterol-rich HDL particles. Future research should focus on narrowing down focus through computational, structural and functional studies to identify the specific molecule or molecules that are responsible for the expected cardioprotective effect of HDL.

References
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22. Kim DS, Marsillach J, Furlong CE, Jarvik GP. Pharmacogenetics of paraoxonase activity: elucidating the role of high-density lipoprotein in disease. Pharmacogenomics 2013; 14: 1495–1515.

The authors
Daniel Seung Kim^1–3† BS; Patrick M. Hutchins^4† PhD; Gail P. Jarvik^1,2 MD, PhD
¹Department of Genome Sciences, University of Washington, Seattle, WA, USA
²Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA, USA
³Department of Biostatistics, University of Washington, Seattle, WA, USA
⁴Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Seattle, WA, USA
^†Authors contributed equally to this work
*Corresponding author
E-mail: pair@u.washington.edu

Acknowledgement
_{DSK is supported in part by 1F31MH101905-01 and a Markey Foundation Award. PMH is supported by a Cardiovascular Fellowship Training Grant (NIH T32HL007828). Work on the CLEAR study referenced within was supported by National Institutes of Health RO1 HL67406 and a State of Washington Life Sciences Discovery Award (265508) to the Northwest Institute of Genetic Medicine.}